Detection of transmembrane potentials by optical methods

ABSTRACT

Methods and compositions are provided for detecting changes in membrane potential in membranes biological systems. In one aspect, the method comprises;
         a) providing a living cell with a first reagent comprising a charged hydrophobic molecule which is typically a fluorescence resonance energy transfer (FRET) acceptor or donor, or is a quencher and is capable of redistributing within the membrane of a biological membrane in response to changes in the potential across the membrane;   b) providing the cell with a second reagent that can label the first face or the second face of a biological membrane within the cell;   c) detecting light emission from the first reagent or the second reagent.       

     One aspect of this method involves monitoring membrane potential changes in subcellular organelle membranes in a living cells. 
     Another aspect of the invention is the use of certain embodiments of the method for the screening of test chemicals for activity to modulate the activity of a target ion channel. 
     Another aspect of the present invention is a transgenic organism comprising a first reagent that comprises a charged hydrophobic fluorescent molecule, and a second reagent comprising a bioluminescent or naturally fluorescent protein.

This application is a continuation application of U.S. application Ser.No. 09/967,772, filed Sep. 28, 2001, now pending, which claims priorityunder 35 U.S.C. § 120 to U.S. application Ser. No. 09/459,956, filedDec. 13, 1999, now issued as U.S. Pat. No. 6,342,379, which claimspriority under 35 U.S.C. § 120 to U.S. application Ser. No. 08/765,860,filed May 8, 1997, now issued as U.S. Pat. No. 6,107,066, which enteredthe national stage from international application No. PCT/US96/09652,filed Jun. 6, 1996, which claims priority under 35 U.S.C. § 120 to U.S.application Ser. No. 08/481,977, filed Jun. 7, 1995, now issued as U.S.Pat. No. 5,661,035, the disclosure of each is incorporated by referencein the disclosure of this application.

This invention was made with Government support under Grant No. RO1NS27177-07, awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to compositions and opticalmethods for determining transmembrane potentials across biologicalmembranes of living cells.

BACKGROUND OF THE INVENTION

Fluorescence detection and imaging of cellular electrical activity is atechnique of great importance and potential (Grinvald, A., Frostig, R.D., Lieke, E., and Hildesheim, R. 1988. Optical imaging of neuronalactivity. Physiol. Rev. 68:1285–1366; Salzberg, B. M. 1983. Opticalrecording of electrical activity in neurons using molecular probes. InCurrent Methods in Cellular Neurobiology. J. L. Barker, editor. Wiley,N.Y. 139–187; Cohen, L. B. and S. Lesher. 1985. Optical monitoring ofmembrane potential: methods of multisite optical measurement. In OpticalMethods in Cell Physiology. P. de Weer and B. M. Salzberg, editors.Wiley, N.Y. 71–99).

Mechanisms for optical sensing of membrane potential have traditionallybeen divided into two classes:

(1) sensitive but slow redistribution of permeant ions from theextracellular medium into the cell, and

(2) fast but small perturbations of relatively impermeable dyes attachedto one face of the plasma membrane. see, Loew, L. M., “How to choose apotentiometric membrane probe”, In Spectroscopic Membrane Probes. L. M.Loew, ed., 139–151 (1988) (CRC Press, Boca Raton); Loew, L. M.,“Potentiometric membrane dyes”, In Fluorescent and Luminescent Probesfor Biological Activity. W. T. Mason, ed., 150–160 (1993) (AcademicPress, San Diego).

The permeant ions are sensitive because the ratio of theirconcentrations between the inside and outside of the cell can change byup to the Nernstian limit of 10-fold for a 60 mV change in transmembranepotential. However, their responses are slow because to establish newequilibria, ions must diffuse through unstirred layers in each aqueousphase and the low-dielectric-constant interior of the plasma membrane.Moreover, such dyes distribute into all available hydrophobic bindingsites indiscriminately. Therefore, selectivity between cell types isdifficult. Also, any additions of hydrophobic proteins or reagents tothe external solution, or changes in exposure to hydrophobic surfaces,are prone to cause artifacts. These indicators also fail to give anyshift in fluorescence wavelengths or ratiometric output. Suchdual-wavelength readouts are useful in avoiding artifacts due tovariations in dye concentration, path length, cell number, sourcebrightness, and detection efficiency.

By contrast, the impermeable dyes can respond very quickly because theyneed little or no translocation. However, they are insensitive becausethey sense the electric field with only a part of a unit charge movingless than the length of the molecule, which in turn is only a smallfraction of the distance across the membrane. Furthermore, a significantfraction of the total dye signal comes from molecules that sit onirrelevant membranes or cells and that dilute the signal from the fewcorrectly placed molecules.

In view of the above drawbacks, methods and compositions are neededwhich are sensitive to small variations in transmembrane potentials andcan respond both to rapid, preferably on a millisecond timescale, andsustained membrane potential changes. Also needed are methods andcompositions that are less susceptible to the effects of changes inexternal solution composition, more capable of selectively monitoringmembranes of specific cell types, and within intracellular organellesand providing a ratiometric fluorescence signal.

Such methods require effective methods of discriminating measurementsfrom within defined cell populations, or subcellular structures. Thisinvention fulfils this and related needs.

SUMMARY OF THE INVENTION

Methods and compositions are provided for detecting changes in membranepotential in biological systems. One aspect of the detection methodcomprises;

a) providing a living cell with a first reagent comprising a chargedhydrophobic molecule. Typically the molecule is a fluorescence resonanceenergy transfer (FRET) acceptor or donor, or is a quencher and iscapable of redistributing within the membrane of a biological membranein response to changes in the potential across the membrane;

b) providing the cell with a second reagent that can label the firstface or the second face of a biological membrane within the cell. In oneaspect, the second reagent can redistribute from the membrane to othersites in response to changes in the potential of the membrane. Typicallythe second reagent comprises a luminescent or fluorescent componentcapable of undergoing energy transfer with the first reagent orquenching light emission of the first reagent;

c) detecting light emission from the first reagent or the secondreagent.

In one aspect of this method, the cell is exposed to excitation light atappropriate wavelengths and the degree of energy transfer between thefirst and second reagents determined. In one contemplated version ofthis method the excitation light is used to confocally illuminate thecell, thereby providing enhanced spatial resolution. In another aspectthis is achieved via the use of two photon excitation.

In another aspect of this method, the cell is exposed to a luminescentor bioluminescent substrate for the luminescent component resulting inlight emission from the luminescent component. The degree of energytransfer between the first and second reagent may then be determined bymeasuring the emission ratios of the first and second reagents, withoutthe need to provide external illumination.

In one aspect of this method, light emission of the first reagent or thesecond reagent is dependent on the membrane potential across themembrane.

In another aspect of this method, the efficiency of energy transfer fromthe first reagent to the second reagent is dependent on the voltagepotential across the membrane.

In another aspect, the cell additionally comprises an ion channel,receptor, transporter or membrane pore-forming agent that acts to setthe membrane potential to a specific value.

Another aspect of the invention involves a method of monitoringsubcellular organelle membrane potentials in a living cell comprising;

-   -   1) providing a living cell with a first reagent, comprising a        hydrophobic, charged fluorescent molecule, and    -   2) providing the living cell with a second reagent comprising a        luminescent or fluorescent component, wherein the luminescent or        fluorescent component is targetable to the subcellular membrane,        and wherein the second reagent undergoes energy transfer with        said first reagent or quenches light emission of the first        reagent.

In one aspect of this method the second reagent is targetable to thesubcellular membrane through fusion to a protein or peptide thatcontains a targeting or localization sequence(s). Preferred localizationsequences provide for specific localization of the protein to thedefined location, with minimal accumulation of the reagent in otherbiological membranes.

Another aspect of the present invention is a transgenic organismcomprising a first reagent that comprises a charged hydrophobicfluorescent molecule, and a second reagent comprising a bioluminescentor naturally fluorescent protein. The bioluminescent or naturallyfluorescent protein is typically expressed within the transgenicorganism and targetable to a cellular membrane. A second reagentprovided to the transgenic organism undergoes energy transfer with thefirst reagent or quenches light emission of said first reagent Suchtransgenic organisms may be used in whole animal studies to monitor drugeffects on neuronal activity in vivo or to understand disease states inappropriate model systems.

Another aspect of the invention is a method of screening test chemicalsfor activity to modulate a target ion channel, involving, providing aliving cell comprising a target ion channel, and a membrane potentialmodulator wherein the membrane potential modulator sets the restingmembrane potential to a predefined value between about −150 mV and +100mV. After contact of the living cell with a test chemical, the membranepotential across the cellular membrane is detected.

These methods are particularly suited for measuring the effects of testchemicals on rapidly inactivated or voltage dependent ion channels. Theinvention works in one aspect by creating a cell with a defined membranepotential through the expression and regulation of the membranepotential modulator. By providing cells with defined membrane potentialsthe invention provides for stable assays for rapidly inactivating ionchannels, and provides a simple and convenient method of activatingvoltage dependent ion channels by appropriate regulation of the activityof the membrane potential modulator.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood with reference to theaccompanying drawings, in which:

FIGS. 1A and 1B illustrate a scheme of the voltage-sensitive FRETmechanism;

FIG. 2 illustrates normalized excitation and emission spectra for (A)fluorescein-labeled wheat germ agglutinin (FL-WGA) in Hanks' BalancedSalt Solution (HBSS), (B) (1,3-dihexyl-2-thiobarbiturate)trimethineoxonol [DiSBA-C₆-(3)] in octanol, and (C) TR-WGA in HBSS;

FIG. 3 illustrates displacement currents of 2.3 M DiSBA-C₆-(3) inL-M(TK⁻) cells at 20 C;

FIG. 4 illustrates voltage dependence of DiSBA-C₆-(3) moved during thedisplacement and tailcurrents for step voltage changes from a −30 mVholding potential;

FIG. 5 illustrates voltage dependence of DiSBA-C₆-(3) displacementcurrent time constants in L-M(TK⁻)cells for the same data shown in FIG.4;

FIG. 6 illustrates simultaneous fluorescence changes of theFL-WGA/DiSBA-C₄-(3) pair in response to 4 depolarizations from −70 mV of40, 80, 120, and 160 mV in a L-M(TK⁻) cell at 20 C, with the singlewavelength fluorescence emission traces of DiSBA-C₄-(3) and FL-WGA beingshown in panels A and B, respectively, and the FL-WGA/DiSBA-C₄-(3) ratiodisplayed in (C);

FIG. 7 illustrates the time course of the fluorescence change of theFL-WGA/DiSBA-C₁₀-(3)pair in response to a 100 mV depolarization from −70mV;

FIG. 8 illustrates a single sweep trace of fluorescence ratio changesfrom the FL-WGA/DiSBA-C₄-(3) pair in beating neonatal cardiac myocytes,with the top trace (A) showing the FL-WGA channel, (B) the longerwavelength oxonol channel and (C) the FL-WGA/oxonol ratio, in whichmotion artifacts are significantly reduced; and

FIG. 9 illustrates the fluorescence changes of the FL-WGA/DiSBA-C₆-(3)pair in a voltage clamped astrocytoma cell, the top trace (A) being theDiSBA-C₆-(3) emission, (B) the FL-WGA fluorescence signal and (C) theFL-WGA/oxonol ratio.

FIG. 10 shows the synthesis of a fluorescent tetraaryl borate.

FIG. 11 shows a synthesis of an asymmetric oxonol and its linkage to asecond reagent.

FIG. 12 shows possible linkage points (X) of oxonols to a secondreagent.

FIG. 13 shows a synthesis of Di-SBA-C₆-(3).

FIG. 14 shows the synthesis of a bifunctional linker.

FIGS. 15 and 16 show the synthesis of an asymmetric oxonol with a linkersuitable for attachment to a second reagent.

FIG. 17 shows FRET between Cou-PE, a conjugate of a6-chloro-7-hydroxycoumarin to dimyristoylphosphatidylethanolamine, asFRET donor, to a bis-(1,3-dihexyl-2-thiobarbiturate)-trimethineoxonol inan astrocytoma cell.

FIG. 18 shows FRET between DMPE-glycine-coumarin (Cou-PE) as FRET donorto a bis-(1,3-dihexyl-2-thiobarbiturate)-trimethineoxonol in L-cells.

FIG. 19 shows FRET between DMPE-glycine-coumarin (Cou-PE) as FRET donorto a bis-(1,3-dihexyl-2-thiobarbiturate)-trimethineoxonol incardiomyocytes measured by ratio output.

FIG. 20 shows representative fluorescent phosphatidylethanolamineconjugates that function as FRET donors to the oxonols. The structureson the left depict representative fluorophores and X denotes the site ofattachment of the phosphatidylethanolamine (PE). The structure (PE-R) onthe right shows a phosphatidylethanolamine where R denotes a fluorophoreattached to the amine of the ethanolamine.

FIG. 21 shows (A) emission spectrum of the Cou-PE; (B) the excitationspectrum of DiSBA-C₆-(5); and (C) the emission spectrum of DiSBA-C₆-(5).

FIG. 22 shows the speed of DiSBA-C₆(5) translocation in response to a100 mV depolarization step, using FRET from asymmetrically labeledCou-PE.

FIG. 23 shows the spectra of [Tb(Salen)₂]⁻¹ (top) and [Eu(Salen)₂]⁻¹(bottom), both as piperidinium salts dissolved in acetonitrile.

FIG. 24A shows a schematic representation of exogenous fluorescent lipiddonors bound to the outer membrane of a bimolecular leaflet. B shows thestructure of bis (1,3-dialkyl-2-thiobarbiturate), one of the trimethineoxonol acceptors of the present invention. C shows a schematic view of apossible arrangement of the GFP/oxonol sensor with a GFP donor locatedon one surface of the biological membrane. Upon depolarization, theoxonol acceptors move to the lower membrane surface resulting inenhanced FRET due to the decrease in mean distance between the donor andacceptor.

FIG. 25. Shows an example ratiometric voltage-sensitive FRET signal in avoltage clamped single cell. In this example the cell was held at −40 mVand hyperpolarized to −80 mV at approximately 300 ms per time point. Atapproximately 1600 ms, the cell was depolarized 100 mV to +20 mV. Theoxonol and GFP fluorescence intensities synchronously change in oppositedirections. In this case, the direction of the fluorescence changesindicate that the GFP donor resides on the inner plasma membraneleaflet. Note how the calculation of the emission ratio eliminates thenoise in the GFP and oxonol signals. The voltage clamp stimulus protocolis shown below.

FIG. 26. Shows membrane potential assays using GFP/oxonol FRET voltagesensors performed in a 96 well plate. The additions of buffer containingvarious potassium concentrations were added using the VIPR™ plate readerbetween 12 and 15 seconds, as indicated by the arrow to RBL cellsexpressing the lyn-sapphire GFP fusion protein. (add patent citation).The cells were loaded with 4 uM DiSBAC₆. The traces show the oxonol toGFP ratio normalized to the starting ratio prior to high K+ stimulus atvarious extracellular K concentrations.

FIG. 27. Shows a dose response of barium on endogenously expressed IRK1and the high K+ response as determined using RBL cells expressing thelyn-sapphire GFP fusion, and loaded with 10 uM DiSBAC₄. The highpotassium and normal sodium containing buffer controls in the absence ofantagonist are shown to the left and right of the curve, respectively.The error bars are +/− standard deviation of 5 wells at eachconcentration.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The following definitions are set forth to illustrate and define themeaning and scope of the various terms used to describe the inventionherein.

Generally, the nomenclature used herein and many of the fluorescence,computer, detection, chemistry and laboratory procedures described beloware those well known and commonly employed in the art Standardtechniques are usually used for chemical synthesis, fluorescence,optics, molecular biology, computer software and integration. Generally,chemical reactions, cell assays and enzymatic reactions are performedaccording to the manufacturer's specifications where appropriate. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references.(Lakowicz, J. R. Topics in Fluorescence Spectroscopy, (3 volumes) NewYork: Plenum Press (1991), and Lakowicz, J. R. Emerging applications offluorescence spectroscopy to cellular imaging: lifetime imaging,metal-ligand probes, multi-photon excitation and light quenching.Scanning Microsc Suppl Vol. 10 (1996) pages 213–24, for fluorescencetechniques; Sambrook et al. Molecular Cloning: A Laboratory Manual,2^(nd) ed. (1989) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., for molecular biology methods; Cells: A Laboratory Manual,1^(st) edition (1998) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., for cell biology methods; Optics Guide 5 Melles Griot®Irvine, Calif., and Optical Waveguide Theory, Snyder & Love published byChapman & Hall for general optical methods, which are incorporatedherein by reference and which are provided throughout this document).

As employed throughout the disclosure, the following terms, unlessotherwise indicated, shall be understood to have the following meanings:

The term “hydrocarbyl” shall refer to an organic radical comprised ofcarbon chains to which hydrogen and other elements are attached. Theterm includes alkyl, alkenyl, alkynyl and aryl groups, groups which havea mixture of saturated and unsaturated bonds, carbocyclic rings andincludes combinations of such groups. It may refer to straight chain,branched-chain, cyclic structures or combinations thereof.

The term “alkyl” refers to a branched or straight chain acyclic,monovalent saturated hydrocarbon radical of one to twenty carbon atoms.

The term “alkenyl” refers to an unsaturated hydrocarbon radical whichcontains at least one carbon-carbon double bond and includes straightchain, branched chain and cyclic radicals.

The term “alkynyl” refers to an unsaturated hydrocarbon radical whichcontains at least one carbon-carbon triple bond and includes straightchain, branched chain and cyclic radicals.

The term “lower” referred to herein in connection with organic radicalsor compounds respectively defines such with up to and including six,preferably up to and including four carbon atoms. Such groups may bestraight chain or branched.

The term “heteroalkyl” refers to a branched or straight chain acyclic,monovalent saturated radical of two to forty atoms in the chain in whichat least one of the atoms in the chain is a heteroatom, such as, forexample, oxygen or sulfur.

The term “lower-alkyl” refers to an alkyl radical of one to six carbonatoms. This term is further exemplified by such radicals as methyl,ethyl, n-propyl, isopropyl, isobutyl, sec-butyl, n-butyl and tert-butyl,n-hexyl and 3-methylpentyl.

The term “cycloalkyl” refers to a monovalent saturated carbocyclicradical of three to twelve carbon atoms in the carbocycle.

The term “heterocycloalkyl” refers to a monovalent saturated cyclicradical of one to twelve atoms in the ring, having at least oneheteroatom, such as oxygen or sulfur) within the ring.

The term “alkylene” refers to a fully saturated, cyclic or acyclic,divalent, branched or straight chain hydrocarbon radical of one to fortycarbon atoms. This term is further exemplified by radicals such asmethylene, ethylene, n-propylene, 1-ethylethylene, and n-heptylene.

The term “heteroalkylene” refers to an alkylene radical in which atleast one of the atoms in the chain is a heteroatom.

The term “heterocyclo-diyl” refers to a divalent radical containing aheterocyclic ring. The free valences may both be on the heterocyclicring or one or both may be on alkylene substituents appended onto thering.

The term “lower-alkylene” refers to a fully saturated, acyclic,divalent, branched or straight chain hydrocarbon radical of one to sixcarbon atoms. This term is further exemplified by such radicals asmethylene, ethylene, n-propylene, i-propylene, n-butylene, i-butylene(or 2-methyl propylene), isoamylene (or 3,3 dimethylpropylene),pentylene, and n-hexylene.

The term “cycloalkyl lower-alkyl” refers to a cycloalkyl group appendedto a lower-alkyl radical This term is exemplified by, but not limitedto, groups such as cyclopropylmethyl, cyclopentylmethyl,cyclopentylethyl, and cyclopentylpropyl.

The term “substituted phenyl” refers to a phenyl group which is mono-,di-, tri-, or tetra-substituted, independently, with hydrocarbyl, alkyl,lower-alkyl, cycloalkyl or cycloalkyl-lower alkyl.

The term “aryl” refers to an aromatic monovalent carbocyclic radicalhaving a single ring (e.g., phenyl) or multiple condensed rings (e.g.,naphthyl, anthracenyl), which can optionally be mono-, di-, ortri-substituted, independently, with hydrocarbyl, alkyl, lower-alkyl,cycloalkyl or cycloalkyl lower alkyl.

The term “arylene” refers to an aromatic divalent carbocyclic radical.The open valence positions may be at any position on the ring(s). In thecase of a divalent phenyl radical, they may be ortho, meta or para toeach other.

The term “aralkyl” refers to an aryl group appended to a lower-alkylradical. This term is exemplified by, but not limited to, groups such asbenzyl, 2-phenylethyl and 2-(2-naphthylethyl).

The term “aralkenyl” refers to an aryl group appended to a fullyconjugated alkenyl radical. This term is exemplified by styrenyl (cisand trans) and 1-phenyl butadienyl and 1-naphthyl butadienyl (includingall possible combinations of the Z and E isomers about the doublebonds).

The term “halo” refers to fluoro, bromo, chloro and iodo.

The term “lower-alkylthio” refers to the group R—S—, where R islower-alkyl.

The term “leaving group” means a group capable of being displaced by anucleophile in a chemical reaction, for example halo, alkyl sulfonates(e.g., methanesulfonate), aryl sulfonates, phosphates, sulfonic acid,sulfonic acid salts, imidazolides, N-hydroxy succinimides and the like.

The term “linker” refers to any chemically and biologically compatiblecovalent grouping of atoms which can serve to link together the firstand second reagents of this invention. Generally, preferred linkers havefrom 20 to 40 bonds from end to end, preferably 25 to 30 bonds, and maybe branched or straight chain or contain rings. The bonds may becarbon-carbon or carbon-heteroatom or heteroatom-heteroatom bonds. Thelinkage can be designed to be hydrophobic or hydrophilic. The linkinggroup can contain single and/or double bonds, 0–10 heteroatoms (O, Spreferred), and saturated or aromatic rings. The linking group maycontain groupings such as ester, ether, sulfide, disulfide and the like.

The term “amphiphilic” refers to a molecule having both a hydrophilicand a hydrophobic portion.

The term “bioluminescent protein” refers to a protein capable of causingthe emission of light through the catalysis of a chemical reaction. Theterm includes proteins that catalyze bioluminescent or chemiluminescentreactions, such as those causing the oxidation of luciferins. The term“bioluminescent protein” includes not only bioluminescent proteins thatoccur naturally, but also mutants that exhibit altered spectral orphysical properties.

The term “fluorescent component” refers to a component capable ofabsorbing light and then re-emitting at least some fraction of thatenergy as light over time. The term includes discrete compounds,molecules, naturally fluorescent proteins and marco-molecular complexesor mixtures of fluorescent and non-fluorescent compounds or molecules.The term “fluorescent component” also includes components that exhibitlong lived fluorescence decay such as lanthanide ions and lanthanidecomplexes with organic ligand sensitizers, that absorb light and thenre-emit the energy over milliseconds.

The term “FRET” refers to fluorescence resonance energy transfer. Forthe purposes of this invention, FRET refers to energy transfer processesthat occur between two fluorescent components, a fluorescent componentand a non-fluorescent component, a luminescent component and afluorescent component and a luminescent component with a non-fluorescentcomponent.

The term “gene knockout” as used herein, refers to the targeteddisruption of a gene in vivo with complete loss of function that hasbeen achieved by any transgenic technology familiar to those in the art.In one embodiment, transgenic animals having gene knockouts are those inwhich the target gene has been rendered nonfunctional by an insertiontargeted to the gene to be rendered non-functional by homologousrecombination.

The term “heterologous” refers to a nucleic acid sequence that eitheroriginates from another species or is modified from either its originalform or the form primarily expressed in the cell.

The term “homolog” refers to two sequences or parts thereof, that aregreater than, or equal to 75% identical when optimally aligned using theALIGN program. Homology or sequence identity refers to the following.Two amino acid sequences are homologous if there is a partial orcomplete identity between their sequences. For example, 85% homologymeans that 85% of the amino acids are identical when the two sequencesare aligned for maximum matching. Gaps (in either of the two sequencesbeing matched) are allowed in maximizing matching; gap lengths of 5 orless are preferred with 2 or less being more preferred. Alternativelyand preferably, two protein sequences (or polypeptide sequences derivedfrom them of at least 30 amino acids in length) are homologous, as thisterm is used herein, if they have an alignment score of more than 5 (instandard deviation units) using the program ALIGN with the mutation datamatrix and a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlasof Protein Sequence and Structure, 1972, volume 5, National BiomedicalResearch Foundation, pp. 101–110, and Supplement 2 to this volume, pp.1–10.

The term “luminescent component” refers to a component capable ofabsorbing energy, such as electrical (e.g. Electro-luminescence),chemical (e.g. chemi-luminescence) or acoustic energy and then emittingat least some fraction of that energy as light over time. The term“component” includes discrete compounds, molecules, bioluminescentproteins and marco-molecular complexes or mixtures of luminescent andnon-luminescent compounds or molecules that act to cause the emission oflight.

The term “membrane potential modulator” refers to components capable ofaltering the resting or stimulated membrane potential of a cellular orsubcellular compartment. The term includes discrete compounds, ionchannels, receptors, pore forming proteins or any combination of thesecomponents.

The term “naturally fluorescent protein” refers to a protein capable offorming a highly fluorescent, intrinsic chromophore either through thecyclization and oxidation of internal amino acids within the protein orvia the enzymatic addition of a fluorescent co-factor. The term includeswild-type fluorescent proteins and engineered mutants that exhibitaltered spectral or physical properties. The term does not includeproteins that exhibit weak fluorescence by virtue only of thefluorescence contribution of non-modified tyrosine, tryptophan,histidine and phenylalanine groups within the protein.

The term “Naturally occurring” refers to a component produced by cellsin the absence of artifical genetic or other modifications of thosecells.

The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “targetable” refers to a component that has the ability to belocalized to a specific location under certain conditions. For example,a protein that can exist at two or more locations that has the abilityto translocate to a defined site under some condition(s) is targetableto that site. Common examples include the translocation of proteinkinase C to the plasma membrane upon cellular activation, and thebinding of SH2 domain containing proteins to phosphorylated tyrosineresidues. The term includes components that are persistently associatedwith one specific location or site, under most conditions.

The term “test chemical” refers to a chemical to be tested by one ormore screening method(s) of the invention as a putative modulator. Atest chemical can be any chemical, such as an inorganic chemical, anorganic chemical, a protein, a peptide, a carbohydrate, a lipid, or acombination thereof. Usually, various predetermined concentrations oftest chemicals are used for screening, such as 0.01 micromolar, 1micromolar and 10 micromolar. Test chemical controls can include themeasurement of a signal in the absence of the test compound orcomparison to a compound known to modulate the target.

The term “transformed” refers to a cell into which (or into an ancestorof which) has been introduced, by means of recombinant nucleic acidtechniques, a heterologous nucleic acid molecule.

The term “transgenic” is used to describe an organism that includesexogenous genetic material within all of its cells The term includes anyorganism whose genome has been altered by in vitro manipulation of theearly embryo or fertilized egg or by any transgenic technology to inducea specific gene knockout.

The term “transgene” refers any piece of DNA which is inserted byartifice into a cell, and becomes part of the genome of the organism(i.e., either stably integrated or as a stable extrachromosomal element)which develops from that cell. Such a transgene may include a gene whichis partly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Included within this definition is a transgene created bythe providing of an RNA sequence that is transcribed into DNA and thenincorporated into the genome. The transgenes of the invention includeDNA sequences that encode the fluorescent or bioluminescent protein thatmay be expressed in a transgenic non-human animal.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “comparisonwindow”, “sequence identity”, “percentage identical to a sequence”, and“substantial identity”. A “reference sequence” is a defined sequenceused as a basis for a sequence comparison; a reference sequence may be asubset of a larger sequence, for example, as a segment of a full-lengthcDNA or gene sequence given in a sequence listing such as a SEQ. ID. NO:1, or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 20 nucleotides in length, frequently atleast 25 nucleotides in length, and often at least 50 nucleotides inlength. Since two polynucleotides may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide sequence) that issimilar between the two polynucleotides, and (2) may further comprise asequence that is divergent between the two polynucleotides, sequencecomparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 20 contiguous nucleotide positionswherein a polynucleotide sequence may be compared to a referencesequence of at least 20 contiguous nucleotides and wherein the portionof the polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) of 20 percent or less as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. Optimal alignment ofsequences for aligning a comparison window may be conducted by the localhomology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482,by the homology alignment algorithm of Needleman and Wunsch (1970) J.Mol. Biol. 48: 443, by the search for similarity method of Pearson andLipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected. Theterm “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage identical to a sequence” is calculatedby comparing two optimally aligned sequences over the window ofcomparison, determining the number of positions at which the identicalnucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequencesto yield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparison(i.e., the window size), and multiplying the result by 100 to yield thepercentage of sequence identity. The terms “substantial identity” asused herein denotes a characteristic of a polynucleotide sequence,wherein the polynucleotide comprises a sequence that has at least 30percent sequence identity, preferably at least 50 to 60 percent sequenceidentity, more usually at least 60 percent sequence identity as comparedto a reference sequence over a comparison window of at least 20nucleotide positions, frequently over a window of at least 25–50nucleotides, wherein the percentage of sequence identity is calculatedby comparing the reference sequence to the polynucleotide sequence whichmay include deletions or additions which total 20 percent or less of thereference sequence over the window of comparison.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 30 percentsequence identity, preferably at least 40 percent sequence identity,more preferably at least 50 percent sequence identity, and mostpreferably at least 60 percent sequence identity. Preferably, residuepositions which are not identical differ by conservative amino acidsubstitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.

Since the list of technical and scientific terms cannot be allencompassing, any undefined terms shall be construed to have the samemeaning as is commonly understood by one of skill in the art to whichthis invention belongs. Furthermore, the singular forms “a”, “an” and“the” include plural referents unless the context clearly dictatesotherwise. For example, reference to a “restriction enzyme” or a “highfidelity enzyme” may include mixtures of such enzymes and any otherenzymes fitting the stated criteria, or reference to the method includesreference to one or more methods for obtaining cDNA sequences which willbe known to those skilled in the art or will become known to them uponreading this specification.

Before the present sequences, variants, formulations and methods formaking and using the invention are described, it is to be understoodthat the invention is not to be limited only to the particularsequences, variants, formulations or methods described. The sequences,variants, formulations and methodologies may vary, and the terminologyused herein is for the purpose of describing particular embodiments. Theterminology and definitions are not intended to be limiting since thescope of protection will ultimately depend upon the claims.

The compositions used in the methods of the invention typically comprisetwo reagents. The first reagent comprises a mobile hydrophobic moleculethat rapidly redistributes within the biological membrane in response tochanges in transmembrane potential. Typically the first reagent ischarged and preferably positively charged under the physiologicalconditions within a living cell. This species is referred to as themobile or hydrophobic molecule. The second reagent comprises aluminescent or fluorescent component that is capable of undergoingenergy transfer with the first reagent, typically by donating excitedstate energy to the mobile fluorescent molecule. In the case where thesecond reagent is a fluorescent component, it may also be capableaccepting excited state energy from the mobile fluorescent molecule.

The first and second reagents are spectroscopically complementary toeach other, by which is meant that their spectral characteristics aresuch that excited state energy transfer can occur between them. Eitherreagent can function as the donor or the acceptor, in which case theother reagent is the corresponding complement, i.e., the acceptor ordonor respectively. Both FRET and quenching are highly sensitive to thedistance between the two species. For example, the nonradiativeForster-type quenching observed in FRET varies inversely with the sixthpower of the distance between the donor and acceptor species. Therefore,when the membrane potential changes and the hydrophobic fluorescentmolecule moves either further away from or closer to the second reagent,FRET between the two reagents is either reduced or enhancedsignificantly. Other mechanisms such as electron-transfer, Dexterexchange interaction, paramagnetic quenching, and promoted intersystemcrossing are even shorter-range and require the two reagents to collideor at least come within 1 nm of each other.

The present invention includes voltage assays that derive at least somepart of the measurable signal from non-FRET derived changes in lightemission from either the first or second reagent. Such changes in lightemission can occur as a direct or indirect effect of the transmembranepotential on the fluorescent or luminescent properties of first orsecond reagent. For example, translocation of a fluorescent dye into orout of the lipid bilayer can cause significant alterations in the molarextinction coefficient and quantum yield of the dye that can beexploited to significantly amplify a FRET signal.

Naturally fluorescent proteins have been successfully fused to a rangeof proteins and in some cases these have been demonstrated to provideconformation sensitive changes in the fluorescent properties of thefluorescent protein (for example see PCT publication WO 98/30715). Suchchanges in protein conformation can result in alterations in the opticalproperties of the fluorescent or bioluminescent protein that can be usedto measure a membrane potential change. Significantly larger and morespecific signal changes in response to a given transmembrane change mayfurther be provided by combining the approach with the use of FRET to asecond fluorophore.

Previously reported voltage-sensitive fluorescent indicators operatingby potential driven redistribution of the fluorophore across themembrane had response times of >100 ms, often as long as minutes. Oneaspect of the present invention provides highly fluorescent anionic dyeswhich translocate across the membrane at much faster rates, withexponential time constants typically less than about 10 ms, frequentlyless than 5 ms, most frequently from about 1 to 3 ms and most preferablyless than 1 ms time scale (e.g., 0.1 to 1 ms). These translocation ratesare independent of the presence of the second reagent on theextracellular surface of the membrane. Response times of <1 ms arenecessary for accurate measurement of single action potentials inindividual neurons and are obtained with some of the dyes describedherein (e.g., hexyl-substituted pentamethineoxonol, diSBA-C₆-(5)). Otherdyes described herein have response times in the 2–5 ms range, which arefast enough to monitor voltage changes in heart and smooth muscle, manysynaptic potentials in single neurons, and the average firing activityin populations of neurons (for example, mapping the electrical responsesof different regions of the central nervous system to sensory inputs).

The indicators of the present invention are able to follow both rapidand slower voltage changes over a time scale of seconds to minutes.

I. First Reagent-Mobile Hydrophobic Molecules

In the compositions and methods of the present invention, the firstreagent comprises a hydrophobic ion (fluorescence donor, acceptor, orquencher) which serves as a voltage sensor and moves within the membranein response to changes in the transmembrane potential. The distributionof hydrophobic ions between the two membrane-aqueous interfaces (theextracellular interface and the intracellular interface, in the case ofthe plasma membrane) is determined by the membrane potential. Cationswill tend to congregate at the negatively charged membrane interface andcorrespondingly, anions will move to the positively charged interface.The inherent sensitivity of the invention is based on the largeinterfacial concentration changes of the mobile ion at physiologicallyrelevant changes in membrane potentials Potentially, a 60 mV changeproduces 10-fold change in the ratio of the anion concentrations at therespective interfaces. The methods of this invention couple this changein interfacial concentration to an efficient fluorescent readout thusproviding a sensitive method of detecting changes in transmembranepotential. The speed of the fluorescence change is dependent on themembrane translocation rate of the hydrophobic ion.

Preferably, the mobile ions that translocate across the biologicalmembrane are hydrophobic in order to bind strongly to the membrane andtranslocate rapidly across it in response to changes in transmembranepotential. Preferably, the ion will have a single charge which will bedelocalized across a significant portion of the dye, preferably theentire dye. Delocalization of the charge reduces the Born chargingenergy (inversely proportional to anion radius) required to move acharged molecule from a hydrophilic to a hydrophobic environment andfacilitates rapid translocation of ions (Benz, R. 1988. “Structuralrequirement for the rapid movement of charged molecules acrossmembranes”, Biophys. J. 54:25–33). Increasing hydrophobicity minimizesrelease of the bound dye from the plasma membrane and buries the iondeeper into the membrane, which decreases the electrostatic activationenergy for translocation. Polar groups on the ion should be kept to aminimum and shielded as much as possible to disfavor solvation in theheadgroup region of the bilayer. However, hydrophobicity cannot beincreased without limit, because some aqueous solubility is required topermit cellular loading. If necessary, dyes may be loaded with the aidof amphiphilic solubilizing reagents such as beta-cyclodextrin,Pluronics such as Pluronic F-127, or polyethylene glycols such asPEG400, which help solubilize the hydrophobic ions in aqueous solution.

The term “hydrophobic” when used in the context of the hydrophobic ionrefers to a species whose partition coefficient between a physiologicalsaline solution (e.g. HBSS) and octanol is preferably at least about 50,and more preferably at least about 1000. Its adsorption coefficient to aphospholipid bilayer (such as for example a membrane derived from ahuman red blood cell is at least about 100 nm, preferably at least about300 nm (where the membrane is 3 nm). Methods of determining partitioncoefficients and adsorption coefficients are known to those of skill inthe art.

It is generally preferred that the hydrophobic dye be an anionicspecies. Ester groups of biological membranes generate a sizable dipolepotential within the hydrocarbon core of the membrane. This potentialaids anion translocation through the hydrophobic layer but hinderscations. Therefore, where membrane translocation is concerned, anionshave a tremendous inherent speed advantage over cations. For example, itis known that for the isostructural ions tetraphenylphosphonium cationand tetraphenylborate anion, the anion is much more permeable than thecation (Flewelling, R. F. and Hubbell, W. L. 1986. “The membrane dipolepotential in a total membrane potential model”, Biophys. J. 49:541–552).

In one embodiment, the anions should be strongly fluorescent whenadsorbed to the membrane, whereas they should have minimal fluorescencewhen free in aqueous solution. Preferably, the anionic fluorophoresshould be at least four times, and more preferably at least about eighttimes, brighter when adsorbed to the membrane. In the case of thethiobarbiturate oxonols described herein, their fluorescence is about 20fold greater in the membrane than in water. In principle, if the dyebound extremely tightly to the membrane one would not need a high ratioof fluorescence when bound to the membrane to that when free in aqueoussolution; however, because in reality the volume of the membrane is tinyrelative to the aqueous solution and some water solubility is necessaryfor loading of the dye into cells and tissue, it is desirable for thefirst reagent to be at least about four times more strongly fluorescentin a membrane than in aqueous solution.

The anions also should not act as ionophores, especially protonophores,since such behavior may generate sustained leakage currents. Therefore,the protonation pKa of the anion is typically well below 7, preferablybelow 5, more preferably below 3. Red to infra-red wavelengths ofexcitation and emission are preferred to avoid tissue scattering andheme absorbances. Photodynamic damage should be kept as low as possible,probably best by minimizing triplet state formation and the resultinggeneration of singlet oxygen.

Fluorescent hydrophobic ions include polymethine oxonols, tetraarylborates conjugated to fluorophores and fluorescent complexes of rareearth and transition metals.

A. Polymethine Oxonols

The term “polymethine oxonol” refers to molecules comprising twopotentially acidic groups linked via a polymethine chain and possessinga single negative charge delocalized between the two acidic groups. Thepreferred acidic groups are barbiturates or thiobarbiturates. They maybe symmetric or asymmetric, i.e., each of the two (thio)barbiturates maybe the same or different. The symmetric (thio)barbiturate oxonols aredescribed by the conventional shorthand DiBA-C_(n)-(x) andDiSBA-C_(n)-(x), where DiBA refers to the presence of two barbiturates,DiSBA refers to the presence of two thiobarbiturates, C_(n) representsalkyl substituents having n carbon atoms on the nitrogen atoms of the(thio)barbiturates, and x denotes the number of carbon atoms in thepolymethine chain linking the (thio)barbiturates. It has beenunexpectedly found that oxonols with long chain alkyl substituents (e.g.C_(n) greater than hexyl, especially decyl in the pentamethine oxonols)translocate surprisingly rapidly across plasma membranes.

An extremely useful property of these oxonols is that their fluorescenceemission maximum at 560 nm is 20 times brighter when bound to membranesthan in aqueous solution [Rink, T. J., Montecucco, C., Hesketh, T. R.,and Tsien, R. Y. 1980. Lymphocyte membrane potential assessed withfluorescent probes. Biochim. Biophys. Acta 595:15–30]. Furthermore, thenegative charge is delocalized throughout the chromophore with the fourequivalent oxygens containing the majority of the charge. The highelectron affinity of the thiobarbiturate moieties discouragesprotonation, pKa<1, and resists photooxidative bleaching. The fourN-alkyl groups and the thiocarbonyl give the molecule a necessary amountof hydrophobicity needed for tight membrane binding and rapidtranslocation.

Oxonol compounds used in this invention have a general structure ofFormula I.

wherein:

R is independently selected from the group consisting of H, hydrocarbyland heteroalkyl;

X is independently oxygen or sulfur; and

n is an integer from 1 to 3;

and salts thereof.

The oxonol anions are usually loaded as salts with the cation typicallybeing H⁺, alkali metal, substituted ammonium, or pyridinium.

Preferably X is sulfur, i.e., the hydrophobic anion is abis-(1,3-dialkyl-2-thiobarbiturate)-polymethine oxonol or a derivativethereof.

When R is a hydrocarbyl group, it can be independently selected from thegroup consisting of alkyl, aryl, aralkyl, cycloalkyl and cycloalkyllower-alkyl. Typically these groups have from about 2 to about 40 carbonatoms, more preferably, about 5 to about 20 carbon atoms. Aryl groupscan be substituted with hydrocarbyl, alkyl, lower alkyl, heteroalkyl andhalogen groups. Oxonols in which the R groups on a particular(thio)barbiturate moiety are different to each other are specificallycontemplated by this invention and can be prepared from unsymmetricalurea derivatives.

In some embodiments, R is a hydrocarbyl group of the formula:—(CH₂)_(p)(CH═CH—CH₂)_(q)(CH₂)_(r)CH₃wherein:

p is an integer from 1 to about 20 (preferably about 1 to 2);

q is an integer from 1 to about 6, preferably 1 to 2;

the stereochemistry of the double bond(s) may be cis or trans, cis beingpreferred;

r is an integer from 1 to about 20 (preferably about 1 to 3), andp+3q+r+1 40, preferably from about 4 to 20, more preferably about 6 to10.

In another embodiment of the polymethine oxonols, R is a heteroalkylgroup of the formula:—(CH₂)_(x)A_(y)(CH₂)_(z)CH₃,

wherein:

-   -   A is oxygen or sulfur;    -   x is independently an integer from 1 to about 20 (preferably        about from about 10 to 15);    -   y is independently 0 or 1;    -   z is independently an integer from 1 to about 20 (preferably        about 5 to 10);    -   and x+y+z<40, preferably x+y+z=an integer from about 4 to 25,        more preferably about 4 to 10.

In other embodiments, R is a phenyl group independently substituted withup to four substituents selected from the group consisting ofhydrocarbyl, heteroalkyl, halogen and H.

In other embodiments, one of the four R groups incorporates a linker tothe second reagent, as described below.

An oxonol's negative charge is distributed over the entire thechromophore. Bis(thiobarbiturate)trimethineoxonols absorb at 542 nm(ext. coefficient=200,000 M⁻¹ cm⁻¹), emit at 560 nm and have a quantumyield of 0.4 in octanol. An oxonol where R=n-hexyl, DiSBA-C₆-(3),translocates with a time constant ( )<3 ms in voltage clamped mammaliancells. The corresponding decyl compound, DiSBA-C₁₀-(3), translocateswith a time constant <2 ms. The molecular requirement for rapidtranslocation is nicely met with the symmetric oxonols.Bis(thiobarbiturate)pentamethineoxonols absorb at ˜630 nm and emit at˜660 nm. The negative charge is further delocalized in such red-shiftedoxonols. As expected, the translocation rates for the pentamethineoxonols are faster than for the trimethine oxonols. DiSBA-C₄-(5) crossesthe membrane with <3 ms, six times faster than the correspondingtrimethine oxonol. DiSBA-C₆-(5) translocates with ˜0.4 ms at 20.

B. Tetraaryl Borate-Fluorophore Conjugates

Another useful class of fluorescent hydrophobic anions are tetraarylborates having a general structure of Formula II.[(Ar¹)₃B—Ar²—Y—FLU]—  Formula IIwherein:

Ar¹ is an aryl group;

Ar² is a bifunctional arylene group;

B is boron;

Y is oxygen or sulfur; and

FLU is a neutral fluorophore.

Frequently Ar¹ is substituted with one or two electron withdrawinggroups, such as but not limited to CF₃. In selected embodiments, Ar¹ andAr² are optionally substituted phenyl groups as shown below for thestructure of Formula III.

wherein:

-   -   each R′ is independently H, hydrocarbyl, halogen, CF₃ or a        linker group;    -   n is an integer from 0 to 5;

each X is independently H, halogen or CF₃;

m is an integer from 0 to 4;

Y is oxygen or sulfur; and

FLU is a neutral fluorophore.

When R′ is hydrocarbyl, it is typically from 1 to about 40 carbon atoms,preferably 3 to about 20 carbon atoms, more preferably about 5 to 15carbon atoms. Preferably, R′ is a lower alkyl group, more preferably(for ease of synthesis) all the R's are H. When R′ is not hydrocarbyl,it is frequently CF₃ and n=1. In selected embodiments X=F and m=4. X istypically electron-withdrawing to prevent photoinduced electron transferfrom the tetraaryl borate to the fluorophore, which quenches the latter.X=F is most preferred.

1. Synthesis of Tetraaryl Borate-Fluorophore Conjugates

A general synthesis of fluorescent tetraaryl borate anions has beendeveloped and is shown in FIG. 10 for an exemplary fluorescent bimanetetraaryl borate conjugate (identified as Bormane, compound IV in FIG.10).

In general terms, a triaryl borane is reacted with a protected phenoxyor thiophenoxy organometallic reagent, such as, for example, anorganolithium derivative. The protecting group is subsequently removedand the unmasked phenol (or thiophenol) is reacted with a fluorophorebearing a leaving group. Nucleophilic displacement of the leaving groupfollowed by conventional purification of the crude reaction productfurnishes the tetraaryl borate anion conjugated to the fluorophore.Substituents R′ and X are varied by appropriate choice of the startingtriaryl borane and the phenoxy (or thiophenoxy) organometallic. Suitablestarting materials can be obtained from Aldrich Chemical Co.(Milawaukee, Wis.) and other commercial suppliers known to those ofskill in the art. Thus in these species, a fluorophore is conjugated toa functionalized borate core. This general synthetic method allows oneto attach any fluorophore to the borate anion.

2. Neutral Fluorophores

As polar chromophores retard the membrane translocation rate, it ispreferred that the fluorophore conjugated to the tetraaryl borate be aneutral species. For purposes of the present invention, a neutralfluorophore may be defined as a fluorescent molecule which does notcontain charged functional groups. Representative fluorescent moleculesbearing leaving groups and suitable for conjugation are available fromMolecular Probes (Portland, Oreg.), Eastman Kodak (Huntington, Tenn.),Pierce Chemical Co. (Rockville, Md.) and other commercial suppliersknown to those of skill in the art. Alternatively, leaving groups can beintroduced into fluorescent molecules using methods known to those ofskill in the art.

Particularly suitable classes of neutral fluorophores which can beconjugated to the tetraaryl borates for use in accordance with thepresent invention include, but are not limited to, the following:bimanes; bodipys; and coumarins.

Bodipys (i.e., difluoroboradiazaindacenes) may be represented by ageneral structure of Formula IV.

wherein:

each R¹, which may be the same or different, is independently selectedfrom the group consisting of H, lower alkyl, aryl, heteroaromatic,aralkenyl and an alkylene attachment point;

each R², which may be the same or different, is independently selectedfrom the group consisting of H, lower alkyl, phenyl and an alkyleneattachment point.

For the purposes of this disclosure, the term “alkylene attachmentpoint” refers to the group —(CH₂)_(t)— or —(CH₂)_(t)—C(O)— wherein, t isan integer from 1 to 10, and one valence bond is attached to thefluorophore and the other valence bond is attached to the tetraarylborate. Preferably, t=1, i.e. the alkylene attachment point is amethylene group. As will be apparent to one of skill in the art, allfluorophores will possess one attachment point at which they will beconjugated to the tetraaryl borate. Generally, the precursor moleculeused to conjugate the fluorophore to the tetraaryl borate will carry aleaving group at the attachment point Reaction of this precursor with anappropriate nucleophile on the tetraaryl borate (e.g., an amine, hydroxyor thiol), will provide a fluorophore-tetraaryl borate conjugate linkedtogether at the attachment point The term “attachment point” refers morebroadly to a chemical grouping which is appropriate to react with eithera fluorophore or a bifunctional linker to form the fluorescentconjugates and/or linked first and second reagents as disclosed herein.Frequently, these attachment points will carry leaving groups, e.g.,alkyl tosylates, activated esters (anhydrides, N-hydroxysuccinimidylesters and the like) which can react with a nucleophile on the speciesto be conjugated One of skill will recognize that the relativepositioning of leaving group and the nucleophile on the molecules beinglinked to each other can be reversed.

Coumarins and related fluorophores may be represented by structures ofgeneral Formulas V and VI

wherein:

each R³, which may be the same or different, is independently selectedfrom the group consisting of H, halogen, lower alkyl, CN, CF₃, COOR⁵,CON(R⁵)₂, OR⁵, and an attachment point;

R₄ is selected from the group consisting of OR⁵ and N(R⁵)₂;

Z is O, S or NR⁵; and

each R⁵, which may be the same or different, is independently selectedfrom the group consisting of H, lower alkyl and an alkylene attachmentpoint.

Bimanes may be represented by a structure of general Formula VII.

wherein:

each R⁵, which may be the same or different, is independently H, loweralkyl or an alkylene attachment point.

Fluorescent tetraaryl borates with coumarins and bimanes attached havebeen prepared. These fluorescent borates translocate with <3 ms involtage clamped fibroblasts. Synthesis of an exemplary fluorescenttetraryl borate is described in Example V.

C. Fluorophore Complexes with Transition Metals

Lanthanide ions, such as, for example, Tb³⁺ and Eu³⁺, luminesce in thegreen and red regions of the visible spectrum with millisecondlifetimes. The emission is composed of several sharp bands that areindicative of the atomic origin of the excited states. Direct excitationof the ions can be accomplished using deep UV light. Excitation of thelanthanide ions at longer wavelengths is possible when the ions arechelated by absorbing ligands that can transfer excitation energy to theions, which then can luminesce with their characteristic emission as ifthey had been excited directly. Lanthanide complexes of Tb³⁺ and Eu³⁺with absorbing ligands that contribute 4 negative charges, resulting anet charge of −1, may function as mobile ions for the voltage-sensitiveFRET mechanism. The lifetimes of Tb³⁺ and Eu³⁺ are still sufficientlyfast to measure millisecond voltage changes.

This invention also provides such complexes which can function thefluorescent hydrophobic anion (as FRET donors) in the first reagent.Using the ligand bis-(salicylaldehyde)ethylenediamine (Salen)²⁻,[Tb(Salen)₂]⁻¹ and [Eu(Salen)₂]⁻¹ have been made. These complexes absorbmaximally at 350 nm with significant absorbance up to 380 nm andluminesce with the characteristic atomic emission, FIG. 10. The use oflanthanide complexes as donors offers several unique advantages.Scattering, cellular autofluorescence, and emission from directlyexcited acceptors have nanosecond or shorter lifetimes and may berejected by time gating of the emission acquisition (See for exampleMarriott, G., Heidecker, M., Diamandis, E. P., Yan-Marriott, Y. 1994.Time-resolved delayed luminescence image microscopy using an europiumion chelate complex. Biophys. J. 67: 957–965). The elimination of thefast emission reduces the background and gives excellent signal to noiseratios. Another major advantage of using lanthanide chelates as donorsis that the range of FRET is amplified by lateral diffusion in themembrane during the excited state lifetime (Thomas, D. D., Carlsen, W.F., Stryer, L. 1978. Fluorescence energy transfer in the rapid diffusionlimit. Proc. Natl. Acad. Sci. USA 75: 5746–5750). This feature greatlyreduces the need for high concentrations of acceptors to ensureefficient FRET. In addition to reducing the perturbation and stress tothe cellular system from high dye concentrations, the diffusion enhancedFRET will lead to greater voltage sensitivity than is possible in astatic case. Lanthanide chelates can also be used as asymmetricallylabeled donors to mobile acceptors such as the tri and pentamethineoxonols, with the same advantages as discussed above.

Representative lanthanide complexes which may be used as a hydrophobicfluorescent anion are shown in Formulas XI and XII

wherein:

Ln=Tb, Eu, or Sm;

R is independently H, C1–C8 alkyl, C1–C8 cycloalkyl or C1–C4perfluoroalkyl;

X and Y are independently H, F, Cl, Br, I, NO₂, CF₃, lower (C1–C4)alkyl, CN, Ph, O-(lower alkyl), or OPh; or X and Y together are —CH═CH—;and

Z=1,2-ethanediyl, 1,3-propanediyl, 2,3-butanediyl, 1,2-cyclohexanediyl,1,2-cyclopentanediyl, 1,2-cycloheptanediyl, 1,2-phenylenediyl,3-oxa-1,5-pentanediyl, 3-aza-3-(lower alkyl)-1,5-pentanediyl,pyridine-2,6-bis(methylene) or tetrahydrofuran-2,5-bis(methylene).

wherein:

-   Ln=Tb, Eu, or Sm;-   R is independently H, C1–C8 alkyl, C1–C8 cycloalkyl or C1–C4    perfluoroalkyl;-   X′ and Y′ are independently H, F, Cl, Br, I, NO₂, CF₃, lower (C1–C4)    alkyl, CN, Ph, O-(lower alkyl), or OPh; or X′ and Y′ together are    —CH═CH—; and Z′ is independently a valence bond, CR₂,    pyridine-2–6-diyl or tetrahydofuran-2,5-diyl    II. Targetable, Asymmetrically Bound Second Reagents

The second reagent is a luminescent or fluorescent donor, acceptor, orquencher, complementary to the first reagent, the hydrophobic molecule,and is targetable to either the extracellular or intracellular face ofthe biological membrane. Thus, the presence of the second reagent on oneor the other face of the membrane desymmetrizes the membrane. Asdescribed earlier, energy transfer between the first and second reagentprovides an optical readout read out which changes in response tomovement of the hydrophobic within the biological membrane.

As would be immediately apparent to those skilled in the field, thereare numerous molecular species that could function as the luminescent orfluorescent component and serve as the active desymmetrizing agent. Theprimary characteristics for this component are that it is located on oneface of the biological membrane and function in a complementary manner(i.e., as a fluorescent donor, acceptor, or quencher) to the hydrophobicion which shuttles back forth across the membrane as the transmembranepotential changes.

Exemplary fluorescent second reagents include fluorescent lectins,fluorescent lipids, fluorescent carbohydrates with hydrophobicsubstituents, naturally fluorescent proteins or homologs thereof,fluorescently labelled antibodies against surface membrane constituents,or xanthenes, cyanines and coumarins with hydrophobic and hydrophilicsubstituents to promote binding to membranes and to prevent permeationthrough membranes.

Exemplary luminescent second reagents include chemi-luminescent,electro-luminescent and bioluminescent compounds. Preferredbioluminescent compounds include bioluminescent proteins such asfirefly, bacterial or click beetle luciferases, aequorins and otherphotoproteins, such as Cypridina luciferase.

A. Fluorescent Lectins

One class of second reagents are lectins carrying a fluorescent label.For purposes of the present invention, a lectin may be defined as asugar binding protein which binds to glycoproteins and glycolipids onthe extracellular face of the plasma membrane. See, Roth, J., “TheLectins: Molecular Probes in Cell Biology and Membrane Research,” Exp.Patholo. (Supp. 3), (Gustav Fischer Verlag, Jena, 1978) Lectins includeConcavalin A; various agglutinins (pea agglutinin, peanut agglutinin,wheat germ agglutinin, and the like); Ricin, A chain and the like. Avariety of lectins are available from Sigma Chemical Co., St. Louis, Mo.

Suitable fluorescent labels for use in fluorescent lectins include, butare not limited to, the following: xanthenes (including fluoresceins,rhodamines and rhodols); bodipys, cyanines, and luminescent transitionmetal complexes. It will be recognized that the fluorescent labelsdescribed below can be used not merely with lectins but with the othersecond reagents described herein. To date, the best results with lectinshave been obtained with fluorescein labeled wheat germ agglutinin(FL-WGA).

1. Xanthenes

One preferred class of fluorescent labels comprise xanthene chromophoreshaving a structure of general Formula VIII or IX.

wherein:

R⁶ is independently selected from the group consisting of H, halogen,lower alkyl, SO₃H and an alkylene attachment point;

R⁷ is selected from the group consisting of H, lower alkyl, an alkyleneattachment point, and R⁸, wherein R⁸ is selected from the groupconsisting of

wherein:

-   each a and a′ is independently selected from the group consisting of    H and an alkylene attachment point;-   G is selected from the group consisting of H, OH, OR⁹, NR⁹R⁹ and an    alkylene attachment point;

T is selected from the group consisting of O, S, C(CH₃)₂ and NR⁹; and

M is selected from the group consisting of O and NR⁹R⁹;

wherein each R⁹, which may be the same or different, is independently Hor hydrocarbyl.

2. Cyanines

Another preferred class of fluorescent labels are cyanine dyes having astructure of general Formula X.

wherein:

-   R¹⁵ is independently selected from the group consisting of H,    halogen, lower alkyl, SO₃H, PO₃H₂, OPO₃H₂, COOH, and an alkylene    attachment point;-   R¹⁶ is selected from the group consisting of H, lower alkyl,    (CH₂)_(j)COOH, (CH₂)_(j)SO₃H, and an alkylene attachment point;    where j is an integer from 1 to 10;

T′ is selected from the group consisting of O, S, C(CH₃)₂, —CH═CH—, andNR¹⁷, where R¹⁷ is H or hydrocarbyl; and n is an integer from 1 to 6.

B. Fluorescent Lipids

Fluorescently labeled amphipathic lipids, in particular phospholipids,have also been successfully employed. For purposes of the presentinvention, an amphipathic lipid may be defined as a molecule with bothhydrophobic and hydrophilic groups that bind to but do not readily crossthe cell membrane. Fluorescently labelled phospholipids are ofparticular value as second reagent.

As defined herein, “phospholipids” include phosphatidic acid (PA), andphosphatidyl glycerols (PG), phosphatidylcholines (PC),phosphatidylethanolamines (PE), phospatidylinositols (PI),phosphatidylserines (PS), and phosphatidyl-choline, serine, inositol,ethanolamine lipid derivatives such as egg phosphatidylcholine (EPC),dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine,dipalmitoyl-phosphatidylethanolamine,distearoylphosphatidylethanolamine, dioleoyl-phosphatidylethanolamine,distearoyl-phosphatidylserine, dilinoleoyl phosphatidylinositol, andmixtures thereof. They may be unsaturated lipids and may be naturallyoccurring or synthetic. The individual phosphatidic acid components maybe symmtrical, i.e. both acyl residues are the same, or they may beunsymmetrical, i.e., the acyl residues may be different.

Fluorescent and luminescent lipids constitute an important class ofmolecules that function as immobile second reagents. They can serve asFRET donors, FRET acceptors, fluorescence quenchers and fluorophoresthat can be quenched by voltage-sensitive first reagents. Wellcharacterized embodiments include their use as FRET donors tovoltage-sensitive oxonols. Specific examples include coumarin labeledphospholipids, such as structures A (CC1-DMPE) and B (CC2-DMPE). Ageneric structure is shown in structure C. Other examples of fluorescentphospholipid that function as second reagents include: fluoresceinlabeled phosphatidylethanolamine (PE), NBD labeled PE, and AMCA-Slabeled PE. Coumarin labeled single chain lipids that contain apermanent positive charge, including structure D1, have also been shownto be useful. The positively charged lipid may have electrostaticadvantages with negatively charged second reagents. An electrostaticattraction between the two reagents may enable the probes to associatein the membrane and result in enhanced voltage-sensitive FRET.Furthermore, these probes have been found to allow longer recording ofoptical FRET signals in leech ganglia. A generic structure offluorescently labeled quartenary ammonium lipid is shown in structureD2. The use of fluorescent lipids as FRET acceptors has also beendemonstrated, for example using an oxonol donor and a Cy5-labeled PEacceptor.

The preferred lipid based second reagents comprise 1) a hydrophobiclipid anchor, 2) a charged, polar, or zwitterionic headgroup, and 3) afluorescent component that is anchored to the membrane surface.

The hydrophobic lipid anchor should be sufficiently long to result instrong binding to the membrane (preferably 14–35 carbon or methyleneunits in length). Alternatively it can be composed of severalproportionately (equivalent) smaller segments (i.e. one C₂₀ chain or twoC₁₀ chains). This is desirable to minimize leakage of dye from themembrane location, which could effect the voltage-sensitivity andincrease fluorescence background.

The headgroup should preferably comprise a permanently charged, polar,or zwitterionic group within the normal physiological pH range (pH 6.5to 7.5) so that the molecule does not significantly translocate to theinner membrane leaflet on the timescale of about an hour. The chargedheadgroup prevents the fluorophore and whole molecule from diffusingacross the low dielectric interior of the membrane. Significant membranetranslocation will increase background fluorescence and decreasevoltage-sensitivity due to the loss of asymmetry across the membrane.Preferred are charged and polar headgroups including phosphate,sulfates, quaternary ammonium groups, sugars and amines.

The fluorophore should be attached to the lipid headgroup and be locatedat the membrane-water interface region to maximize the distance changesbetween donor and acceptor when the mobile reagent I is on theintracellular leaflet. The fluorophore is preferably water-soluble.Preferred fluorophores include coumarins, fluoresceins, bodipys,carbocyanines, indocarbocyanines and styryl dyes.

One skilled in the art could meet these criteria in a variety of ways.First as has been demonstrated in the case of PE, one could use a lipidscaffold that contains the appropriate membrane anchoring hydrophobicityand charged headgroup, and then chemically attach various fluorescentgroups to the headgroup. The phosphate group provides the permanentcharge that inhibits membrane translocation. Since PE has a reactiveamino group at the end of the headgroup, one could react any aminoreactive fluorescent labeling reagent (e.g. any of those described inthe Molecular Probes catalog) with PE to produce appropriate secondreagents. Amino reactive reagents could also be prepared fromchromophores not listed in the Molecular Probes catalog using standardsynthetic chemical protocols. Similarly, one could execute the sameprocedure on any amino lipid as long at the headgroup contains apermanent charge at physiological pH. For example, the quaternaryammonium lipid in structure D could be prepared in such a manner tocontain a terminal amino group that could then be reacted with theaforementioned amino reactive groups. A second approach to prepareappropriate lipids would be to synthesize a fluorescent chargedheadgroup that could then be reacted with a variety of hydrophobicanchors. The three components: fluorophore, headgroup, and lipid anchorcan be assembled in any order to produce the same final product Inaddition to amine reactive fluorescent labeling agents, reagents forfluorescently labeling thiol, alcohol, aldehyde, ketone, caboxylic acidfunctional groups are readily available and could be reacted with alipid scaffold that meets the charge and membrane anchor criteria andalso have these functional groups. Of course fluorophore modificationsthat maintain fluorescence of the chromophore would also producemolecules that can function as second reagents. The appropriatechromophore modification are known to those skilled in the art and havebeen described in part in for example in U.S. Pat. No. 5,741,657 issuedApr. 21, 1998 to Tsien et al.

Long-lived (>100 ns) light-emitting lipids can also function as thestationary second reagent. One example class includes lipids with acharged headgroup capable of chelating metal ions and undergoingphoto-induced long-lived emission. These molecules may be enhanced withan organic sensitizer covalently attached. Example of metal ions thatmay be appropriate include: Eu³⁺, Tb³⁺, Sm³⁺, Dy³⁺, Ru^(2+/3+), andRh²⁺.

Synthesis of Compound D1.

Synthesis of coumarin amine[N-(2-(dimethylamino)ethyl)(6-chloro-2-oxo-7-((phenylmethoxy)methoxy)(2H-chromen-3-yl))formamide]:

241 mg 3-carboxy-6 chloro-7-hydroxy coumarin (1 mmol) dissolved in themixture of 5 ml THF and 5 ml DMF was added 200 ul diisopropylethylamine(DIEA, 1.1 mmol) and 330 mg O—(N-succinium)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TSTU, 1.1 mmol). After stirring at roomtemperature for 20 minutes under nitrogen atmosphere, the reactionmixture was added 400 ul of DIEA and 460 ul benzyl chloromethyl ether(BOM-Cl) and stirred for another 2 hours at room temperature. Theresulted mixture was then pulled high vacuum for 2 hours and 1 ml THF,480 ul 2-dimethyaminoethylamine was added into the solution afterwards.After the reaction mixture was allowed to react at room temperature for1 hour, the crude product was pulled high vacuum again overnight andpurified on a silica gel column (chloroform:methanol=95:5 as eluent).190 mg colorless solid was obtained as product, 44% yield. ¹H NMR(CDCl₃, ppm): 2.32 (d, 6H), 2.56 (t, 2H), 2.16 (t, 2H), 4.76 (s, 2H),5.46 (s, 2H), 7.33 (m, 7H), 7.68 (s, 1H), 8.76 (s, 1H). MS:C₂₂H₂₃O₅N₂Cl, 430.3 calculated; 431.2 (M+1) found.

Synthesis of C18-coumarin[N3-(2-(1,1-dimethyl-1-octadecylammonio)-6-chloro-7-hydroxy-2-oxo-2H-3-chromene-carboxamide]:

43 mg coumarin amine (0.1 mmol) from last step synthesis dissolved indichloromethane and 43 mg 1-iodooctadecane was added into the solution.After the reaction mixture was heated with stirring at 80° C. for 24hours under nitrogen atmosphere, solvent was evaporated and the crudeproduct was purified on a silica gel column (chloroform:methnol=8:2 aseluent). 30 mg yellow color solid was obtained as product, 53% yield. ¹HNMR (CD₃OD, ppm): 0.90 (t, 3H), 1.28 (m, 35H), 3.17 (s, 6H), 3.56 (t,2H), 3.90 (t, 2H), 6.73 (s, 1H), 7.78 (s, 1H), 8.69 (s, 1H). MS:C₃₂H₅₂N₂O₄ClI, 691.4 calculated; 563.5 (M−I⁻) found.

C. Fluorescently Labelled Antibodies

Antibodies directed against surface antigens such as glycolipids ormembrane proteins can also be fluorescently labeled and used as secondreagents. For example, FITC-labeled antibodies against the glycolipidGD3 stain the outer surface of the melanoma cell line M21 and give ratiochanges up to 10%/100 mV using DiSBA-C₆-(3) as the mobile fluorescentanion. Specificity for particular cell types is likely to be easier toachieve with antibodies than with lectins because antibodies can beraised against nearly any surface marker Also, microinjected antibodiescould label sites on the cytoplasmic face of the plasma membrane, wherecarbohydrate binding sites for lectins are absent.

D. Cytochromes

Cytochrome c used as a second reagent has also been found to function asa quencher that binds to the outer plasma membrane surface. Accordingly,another suitable class of second reagent comprises cytochrome c orapocytochrome c, with or without a fluorescent group as previouslydescribed in connection with other second reagents.

E. Fluorescent Carbohydrates

Yet another preferred class of embodiments of the second reagentincludes fluorescently labeled, amphipathic carbohydrates, e.g.,cyclodextrins that selectively and tightly bind to the extracellularplasma membrane surface. Typically, the carbohydrates are functionalizedwith a hydrophobic tail to facilitate intercalation into the membraneand tight membrane binding. The cyclic sugar imparts good watersolubility for cellular loading and prohibits membrane translocation.Another added benefit is that the cyclodextrins aid the loading of theoxonol.

F. Fluorescent Peptides and Proteins

Yet another preferred class of embodiments of the second reagentincludes fluorescently labeled, amphipathic peptides. Typically suchpeptides contain several basic residues such as lysines and arginines tobind electrostatically to negatively charged phospholipid head groups,plus several hydrophobic residues to anchor the peptide to the membrane.Optionally, long-chain alkyl substituents such as N-myristoyl,N-palmitoyl, S-palmitoyl, or C-terminal prenyl groups may providehydrophobicity. The fluorescent label is typically attached via lysineepsilon-amino groups or cysteine sulfhydryl groups.

G. Naturally Fluorescent Proteins

Another preferred class of embodiments of the second reagent includesnaturally fluorescent proteins such as the Green Fluorescent Protein(GFP) of Aequorea victoria (Cubitt, A. B. et al. 1995. Understanding,improving, and using green fluorescent proteins. Trends Biochem. Sci.20: 448–455; Chalfie, M., and Prasher, D. C. U.S. Pat. No. 5,491,084).Because the entire fluorophore and peptide of a naturally fluorescentprotein can be expressed within intact living cells without the additionof other co-factors or fluorophores, voltage sensors comprising suchproteins as the second reagent provide the ability to monitor membranepotential changes within defined cell populations, tissues or in anentire transgenic organism, where other reagents could not penetrate orbe specifically localized. For example, by the use of cell type specificpromoters and subcellular targeting motifs, it is possible toselectively target the second reagent to a discrete location to enablehighly spatially defined measurements.

Endogenously fluorescent proteins have been isolated and cloned from anumber of marine species including the sea pansies Renilla reniformis,R. kollikeri and R. mullerei and from the sea pens Ptilosarcus,Stylatula and Acanthoptilum, as well as from the Pacific Northwestjellyfish, Aequorea victoria; Szent-Gyorgyi et al. (SPIE conference1999), D. C. Prasher et al., Gene, 111:229–233 (1992) and severalspecies of coral (Matz et al. Nature Biotechnology 17 969–973 (1999).These proteins are capable of forming a highly fluorescent, intrinsicchromophore through the cyclization and oxidation of internal aminoacids within the protein that can be spectrally resolved from weaklyfluorescent amino acids such as tryptophan and tyrosine.

Additionally fluorescent proteins have also been observed in otherorganisms, although in most cases these require the addition of someexogenous factor to enable fluorescence development. For example, thecloning and expression of yellow fluorescent protein from Vibriofischeri strain Y-1 has been described by T. O. Baldwin et al.,Biochemistry (1990) 29:5509–15. This protein requires flavins asfluorescent co-factors. The cloning of Peridinin-chlorophyll α bindingprotein from the dinoflagellate Symbiodinium sp. was described by B. J.Morris et al., Plant Molecular Biology, (1994) 24:673:77. One usefulaspect of this protein is that it fluoresces in red. The cloning ofphycobiliproteins from marine cyanobacteria such as Synechococcus, e.g.,phycoerythrin and phycocyanin, is described in S. M. Wilbanks et al., J.Biol. Chem. (1993) 268:1226–35. These proteins require phycobilins asfluorescent co-factors, whose insertion into the proteins involvesauxiliary enzymes. The proteins fluoresce at yellow to red wavelengths.

A variety of mutants of the GFP from Aequorea victoria have been createdthat have distinct spectral properties, improved brightness and enhancedexpression and folding in mammalian cells compared to the native GFP,SEQ. ID. NO: 1 Table 1, (Green Fluorescent Proteins, Chapter 2, pages 19to 47, edited Sullivan and Kay, Academic Press, U.S. Pat. Nos.:5,625,048 to Tsien et al., issued Apr. 29, 1997; 5,777,079 to Tsien etal., issued Jul. 7, 1998; and U.S. Pat. No. 5,804,387 to Cormack et al.,issued Sep. 8, 1998). In many cases these functional engineeredfluorescent proteins have superior spectral properties to wild-typeAequorea GFP and are preferred for use as second reagents in the presentinvention.

TABLE 1 Aequorca Fluorescent Proteins Quantum Yield Relative Common (Φ)& Excitation & Fluorescence Sensitivity To Low pH Mutations Name MolarExtinction Emission Max At 37° C. % max F at pH 6 S65T type S6ST, S72A,Emerald Φ = 0.68 487 100 91 N149K, M153T ε = 57,500 509 I167T F64L,S65T, Φ = 0.58 488 54 43 V163A ε = 42,000 511 F64L,S65T EGFP Φ = 0.60488 20 57 ε = 55,900 507 S65T Φ = 0.64 489 12 56 ε = 52,000 511 Y66Htype F64L, Y66H, P4-3E Φ = 0.27 384 100 N.D. Y145F, V163A ε = 22,000 448F64L, Y66H, Φ = 0.26 383 82 57 Y145F ε = 26,300 447 Y66H, Y145F P4-3 Φ =0.3 382 51 64 ε = 22,300 446 Y66H BFP Φ = 0.24 384 15 59 ε = 21,000 448Y66W type S65A, Y66W, WIC Φ = 0.39 435 100 82 S72A, N146I, ε = 21,200495 M153T, V163A F64L, S65T, WIB Φ = 0.4 434 452 80 71 Y66W, N146I, ε =32,500 476 (505) M153T, V163A Y66W, N1461, hW7 Φ = 0.42 434 452 61 88M153T, V163A ε = 23,900 476 (505) Y66W 436 N.D. ND. 485 T203Y type S65G,S72A, Topaz Φ = 0.60 514 100 14 K79R, T203Y ε = 94,500 527 S65G, V68L,10C Φ = 0.61 514 58 21 S72A, T203Y ε = 83,400 527 S65G, V68L, h10C+ Φ =0.71 516 50 54 Q69K, S72A, ε = 62,000 529 T203Y S65G, S72A, Φ = 0.78 50812 30 T203H ε = 48,500 518 S6SG, S72A Φ = 0.70 512 6 28 T203F ε = 65,500522 T203I type T2031, S72A, Sapphire Φ = 0.64 395 100 90 Y145F ε =29,000 511 T2031 H9 Φ = 0.6 395 13 80 T202F ε = 20,000 511

Non Aequorea fluorescent proteins, for example Anthozoan fluorescentproteins, and functional engineered mutants thereof, are also suitablefor use in the present invention including those shown in Table 2 below.

TABLE 2 Anthozoa Fluorescent Proteins Quantum Yield Protein (Φ) &Excitation & Relative Species Name Molar Extinction Emission MaxBrightness SEQ. ID. NO. Anemonia amFP486 Φ = 0.24 458 0.43 SEQ. ID. NO:2 mojano ε = 40,000 486 Zoanthus sp zFP506 Φ = 0.63 496, 506 1.02 SEQ.ID. NO: 3 ε = 35,600 zFP538 Φ = 0.42 ε = 20,200 528, 538 0.38 SEQ. ID.NO: 4 Discosoma dsFP483 Φ = 0.46 443 0.5 SEQ. ID. NO: 5 striata ε =23,900 483 Discosoma sp drFP583 Φ = 0.23 558 0.24 SEQ. ID. NO: 6 “red” ε= 22,500 583 Clavularia sp CFP484 Φ = 0.48 456 0.77 SEQ. ID. NO: 7 ε =35,300 484H. Bioluminescent Proteins

Preferred luminescent components include chemi-luminescent,electro-luminescent and bioluminescent compounds. Preferredbioluminescent components include bioluminescent proteins such asfirefly, bacterial or click beetle luciferases, aequorins and otherphotoproteins, for example as described in U.S. Pat. No. 5,221,623,issued Jun. 22, 1989 to Thompson et al., and U.S. Pat. No. 5,683,888issued Nov. 4, 1997 to Campbell, 5,674,713 issued Sep. 7, 1997 to DeLucaet al., U.S. Pat. No. 5,650,289 issued Jul. 22, 1997 to Wood and U.S.Pat. No. 5,843,746 issued Dec. 1, 1998 to Tatsumi et al.

Particularly preferred are bioluminescent proteins isolated from theostracod Cypridina (or Vargula) hilgendorfii. (Johnson and Shimomura,(1978) Methods Enzymol 57 331–364, Thompson, Nagata & Tsuji (1989) Proc.Natl. Acad. Sci. USA 86, 6567–6571).

Beyond the availability of bioluminescent proteins (luciferases)isolated directly from the light organs of beetles, cDNAS encodingluciferases of several beetle species (including, among others, theluciferase of P. pyralis (firefly), the four luciferase isozymes of P.plagiophthalamus (click beetle), the luciferase of L. cruciata (firefly)and the luciferase of L. lateralis)(deWet et al., Molec. Cell. Biol. 7,725–737 (1987); Masuda et al., Gene 77, 265–270 (1989); Wood et al.,Science 244, 700–702 (1989); European Patent Application Publication No.0 353 464) are available. Further, the cDNAs encoding luciferases of anyother beetle species, which make bioluminescent proteins, are readilyobtainable by the skilled using known techniques (de Wet et al. Meth.Enzymol. 133, 3–14 (1986); Wood et al, Science 244, 700–702 (1989).

Bioluminescent light-emitting systems have been known and isolated frommany luminescent organisms, including certain bacteria, protozoa,coelenterates, molluscs, fish, millipedes, flies, fungi, worms,crustaceans, and beetles, particularly the fireflies of the generaPhotinus, Photuris, and Luciola and click beetles of genus Pyrophorus.

In many of these organisms, enzymatically catalyzed oxido-reductionstake place in which the free energy change is utilized to excite amolecule to a high energy state. Then, when the excited moleculespontaneously returns to the ground state, visible light is emitted.This emitted light is called “bioluminescence.” Typically one quantum oflight is emitted for each molecule of substrate oxidized (which isgenerically referred to as a luciferin). The electronically excitedstate of the oxidized substrate is a state that is characteristic of theluciferase-luciferin reaction of a bioluminescent protein; the color(and, therefore, the energy) of the light emitted upon return of theoxidized substrate to the ground state is determined by the enzyme (orluciferase).

Most firefly and click beetle luciferases are ATP and magnesiumdependent and require oxygen for light production. Typically lightemission from these enzymes exhibits a rapid burst in intensity followedby a rapid decrease in the first few seconds, followed by asignificantly slower sustained light emission. Relatively sustainedlight output at high rates has been accomplished in these systems byinclusion of coenzyme A, dithiothreitol and other reducing agents thatreduce product inhibition and slows inactivation of the luciferase thatoccurs during catalysis of the light producing reaction, as described inU.S. Pat. No. 5,641,641, issued Jun. 24, 1997, and U.S. Pat. No.5,650,289, issued Jul. 22, 1997. Such stable light emitting systems arepreferred for use in the present system.

Particularly preferred bioluminescent proteins are those derived fromthe ostracod Cypridina (or Vargula) hilgendorfii. The Cypridinaluciferase (GenBank accession no. U89490) uses no cofactors other thanwater and oxygen, and its luminescent reaction proceeds optimally at pH7.2 and physiological salt concentrations, (Shimomura, O., Johnson, F.H. and Saiga, Y. (1961) J. Cell. Comp. Physiol. 58 113–124). Bycomparison, firefly luciferase has optimal activity at low ionicstrength, alkaline pH and reducing conditions, that are typically quitedifferent to those usually found within mammalian cells. BecauseCypridina luciferase has a turnover number of 1600 min⁻¹ and a quantumyield of 0.29, (Shimomura, O. & Johnson, F. H. and Masugi, T. (1969)Science 164 1299–1300.Shimomura, O. & Johnson, F. H. (1970) Photochem.Photobiol. 12 291–295), the Cypridina luciferase produces a specificphoton flux exceeding that of the optimized firefly system by a factorof at least 50(Miesenbock and Rothman, Proc. Natl. Acad. Sci. USA (1997)94 3402–3407.

The ready availability of cDNAs encoding various bioluminescent proteinsmakes possible their use in the voltage assays of the present invention.By coupling energy transfer from the bioluminescent protein to theacceptor fluorophore which is mobile within a biological membrane thebioluminescent proteins can provide highly specific and sensitivevoltage measurements.

III. Targeting to Biological Membranes

Typically the fluorescent and bioluminescent components additionallycomprise a localization or targeting sequence to direct or sort thecomponent to a particular face of a biological membrane or subcellularorganelle. Preferred localization sequences provide for highly specificlocalization of the protein, with minimal accumulation in otherbiological membranes. Example localization sequences that directproteins to specific subcellular structures are shown below in Table 3.

TABLE 3 Protein Localization Sequences Preferred Location Sequence SEQ.ID. NO. Orientation Nuclear (Import) PPKKKRKV SEQ. ID. NO. 8 N-terminalEndoplasmic reticulum MSFVSLLLVGILFWATGAENLTK SEQ. ID. NO. 9 N-terminal(Import) CEVFN Endoplasmic reticulum KDEL SEQ. ID. NO. 10 C-terminal,(Retention) KKAA SEQ. ID. NO. 11 Peroxisome (Import) SKL SEQ. ID. NO. 12C-terminal Mitochondrial MLSLRNSIRFEKPATRTLCSSRYLL SEQ. ID. NO. 13N-terminal (Inner membrane) Mitochondrial MLRTSSLFTRRVQPSLFRNILRLQ SEQ.ID. NO. 14 N-terminal (Outer membrane) ST Plasma membraneMGCIKSKRKDNLNDDGVDMKT SEQ. ID. NO: 15 N-terminal (Cytosolic face) Plasmamembrane KKKKKKKSKTKCVIM SEQ. ID. NO: 16 C-terminal (Cytosolic face)

Other examples of localization signals are described in for example, in“Protein Targeting”, chapter 35 of Stryer, L., Biochemistry (4th ed.).W. H. Freeman, 1995 and Chapter 12 (pages 551–598) of Molecular Biologyof the Cell, Alberts et al. third edition, (1994) Garland PublishingInc. In some cases, correct localization requires a number of discretelocalization motifs to provide for correct sorting by the cellularmachinery. For example, correct sorting of proteins to the extracellularface of the plasma membrane requires an N-terminal signal sequence and aC-terminal GPI anchor or transmembrane domain.

Localization sequences in general can be located almost anywhere in theamino acid sequence of the protein. In some cases the localizationsequence can be split into two blocks separated from each other by avariable number of amino acids. The creation of such constructs viastandard recombinant DNA approaches is well known in the art, as forexample described in Maniatis, et al., Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y., 1989).

Localization of the second reagent can also be achieved by fusion to aprotein with a defined pattern of spatial distribution in a given celltype. In this case, the specific localization sequences need not bedefined. Because such fusions to fluorescent or bioluminescent proteinscan be directly visualized by microscopy it is possible to routinelytest previously unknown sequences and determine their utility in theassay.

Another class of localization sequences includes targetable sequencesthat enable conditional binding via an interaction domain to a specificlocation in the cell. Examples of include protein-protein interactiondomains such as SH2, SH3, PDZ, 14,3,3 and PTB domains, protein-DNAdomains such as zinc finger motifs, and protein-lipid interactiondomains such as PH, Ca²⁺/lipid binding domains. Other interactiondomains are described in for example, the database of interactingproteins available on the web at http://www.doe-mbi.ucla.edu. Anadvantage of this approach is that extremely specific patterns oflocalization can be achieved that correspond in vivo to the functionalcompartmentation of proteins within a cell. Such functionalcompartmentation of proteins can play significant roles in specializedcell types such as neurons that have unique cellular architectures.

The approach is generally applicable to any interaction domain. Thecreation of such constructs via standard recombinant DNA approaches iswell known in the art, as for example described in Maniatis, et al.,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y., 1989).

The addition of the domain to the naturally fluorescent or luminescentprotein provides for the potential regulation of binding to thesubcellular site in response to a defined activation signal. In certaincircumstances this can be used to enhance the sensitivity and orspecificity of the fluorescence changes if cellular activation alsoregulates membrane potential. For example, creation of a fusion proteincomprising the bioluminescent or fluorescent protein to a receptor orion channel directly coupled to the voltage change could augment thefluorescence response if stimulation of the cell also resulted in achange in the cellular location of the fusion protein.

IV Transfection and Expression of Reagents

Nucleic acids used to transfect cells with sequences coding forexpression of the polypeptide of interest generally will typically be inthe form of an expression vector including expression control sequencesoperatively linked to a nucleotide sequence coding for expression of thepolypeptide. As used, the term “nucleotide sequence coding forexpression of” a polypeptide refers to a sequence that, upontranscription and translation of mRNA, produces the polypeptide. Thiscan include sequences containing, e.g., introns. As used herein, theterm “expression control sequences” refers to nucleic acid sequencesthat regulate the expression of a nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus, expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalsfor introns, maintenance of the correct reading frame of that gene topermit proper translation of the mRNA, and stop codons.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the fluorescent orbioluminescent protein coding sequence, operatively coupled toappropriate localization or targeting domains and appropriatetranscriptional/translational control signals. These methods include invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. (See, for example, the techniquesdescribed in Maniatis, et al., Molecular Cloning A Laboratory Manual,Cold Spring Harbor Laboratory, N.Y., 1989). Many commercially availableexpression vectors are available from a variety of sources includingClontech (Palo Alto, Calif.), Stratagene (San Diego, Calif.) andInvitrogen (San Diego, Calif.) as well as and many other commercialsources.

A contemplated version of the method is to use inducible controllingnucleotide sequences to produce a sudden increase in the expression ofbioluminescent or fluorescent protein e.g., by inducing expression ofthe construct. Example inducible systems include the tetracyclineinducible system first described by Bujard and colleagues (Gossen andBujard (1992) Proc. Natl. Acad. Sci USA 89 5547–5551, Gossen et al.(1995) Science 268 1766–1769) and described in U.S. Pat. No. 5,464,758.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells that arecapable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ method byprocedures well known in the art. Alternatively, MgCl₂ or RbCl can beused. Transformation can also be performed after forming a protoplast ofthe host cell or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also beco-transfected with DNA sequences encoding the fusion polypeptide of theinvention, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein. (Eukaryotic Viral Vectors,Cold Spring Harbor Laboratory, Gluzman ed., 1982). Preferably, aeukaryotic host is utilized as the host cell as described herein.

V. Transgenic Organisms

In another embodiment, the invention provides a transgenic non-humanorganism that expresses a nucleic acid sequence that encodes abioluminescent or fluorescent protein. Because bio-luminescent andnaturally fluorescent proteins can be specifically expressed withinintact living cells, voltage sensors comprising such proteins as thesecond reagent provide the ability to monitor membrane potential changeswithin defined cell populations, tissues or in an entire transgenicorganism.

Such non-human organisms include vertebrates such as rodents, fish suchas Zebrafish, non-human primates and reptiles as well as invertebratessuch as insects, C. elegans etc. Preferred non-human organisms areselected from the rodent family including rat and mouse, most preferablymouse. The transgenic non-human organisms of the invention are producedby introducing transgenes into the germline of the non-human organism.Embryonic target cells at various developmental stages can be used tointroduce transgenes. Different methods are used depending on theorganism and stage of development of the embryonic target cell. Invertebrates, the zygote is the best target for microinjection. In themouse, the male pronucleus reaches the size of approximately 20micrometers in diameter which allows reproducible injection of 1–2 pl ofDNA solution. The use of zygotes as a target for gene transfer has amajor advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal., Proc. Natl. Acad. Sci. USA 82:4438–4442, 1985). As a consequence,all cells of the transgenic non-human animal will carry the incorporatedtransgene. This will in general also be reflected in the efficienttransmission of the transgene to offspring of the founder since 50% ofthe germ cells will harbor the transgene. Microinjection of zygotes isthe preferred method for incorporating transgenes in practicing theinvention.

A transgenic organism can be produced by cross-breeding two chimericorganisms which include exogenous genetic material within cells used inreproduction. Twenty-five percent of the resulting offspring will betransgenic i.e., organisms that include the exogenous genetic materialwithin all of their cells in both alleles. 50% of the resultingorganisms will include the exogenous genetic material within one alleleand 25% will include no exogenous genetic material.

Retroviral infection can also be used to introduce transgene into anon-human organism. In vertebrates, the developing non-human embryo canbe cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retro viral infection (Jaenich, R., Proc.Natl. Acad. Sci USA 73:1260–1264, 1976). Efficient infection of theblastomeres is obtained by enzymatic treatment to remove the zonapellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viralvector system used to introduce the transgene is typically areplication-defective retro virus carrying the transgene (Jahner, etal., Proc. Natl. Acad. Sci. USA 82:6927–6931, 1985; Van der Putten, etal., Proc. Natl. Acad. Sci USA 82:6148–6152, 1985). Transfection iseasily and efficiently obtained by culturing the blastomeres on amonolayer of virus-producing cells (Van der Putten, supra; Stewart, etal., EMBO J. 6:383–388, 1987).

Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (D. Jahner etal., Nature 298:623–628, 1982). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellsthat formed the transgenic nonhuman animal. Further, the founder maycontain various retro viral insertions of the transgene at differentpositions in the genome that generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline, albeit with low efficiency, by intrauterine retro viral infectionof the midgestation embryo (D. Jahner et al., supra).

A third type of target cell for transgene introduction for vertebratesis the embryonic stem cell (ES). ES cells are obtained frompre-implantation embryos cultured in vitro and fused with embryos (M. J.Evans et al. Nature 292: 154–156, 1981; M. O. Bradley et al., Nature309: 255–258, 1984; Gossler, et al., Proc. Natl. Acad. Sci USA 83:9065–9069, 1986; and Robertson et al., Nature 322:445–448, 1986).Transgenes can be efficiently introduced into the ES cells by DNAtransfection or by retro virus-mediated transduction. Such transformedES cells can thereafter be combined with blastocysts from a nonhumananimal. The ES cells thereafter colonize the embryo and contribute tothe germ line of the resulting chimeric animal. (For review seeJaenisch, R., Science 240: 1468–1474, 1988).

As the preceding discussion indicates and would be readily appreciatedby those skilled in the art, a wide variety of known donor/acceptorpairs can be used as first and second reagents. Particularly preferredcombinations include, but are not limited to, the following, in whichthe first-named fluorophore is the donor and the second is the acceptor:fluorescein/bis-thiobarbiturate trimethineoxonol; Naturally FluorescentProtein/bis-thiobarbiturate trimethineoxonol; Naturally FluorescentProtein/bis-thiobarbiturate pentamethineoxonol;coumarin/bis-thiobarbiturate trimethineoxonol;coumarin/bis-thiobarbiturate pentamethineoxonol; bis-thiobarbituratetrimethineoxonol/Texas Red; bis-thiobarbituratetrimethineoxonol/resorufin; bis-thiobarbiturate trimethineoxonol/Cy5;bis-thiobarbiturate trimethineoxonol/bis-thiobarbituratepentamethineoxonol; Texas Red/bis-thiobarbiturate pentamethineoxonol;NBD/bis-thiobarbiturate trimethineoxonol; NBD/bis-thiobarbituratepentamethineoxonol; Luciferase/Naturally fluorescent protein.

VI. Linker Groups Between First and Second Reagent

In some particularly preferred embodiments of the compositions andmethods of the present invention, a linker group is employed between thefirst and second fluorophores. The linker group maintains a certainminimum proximity between the first and second fluorophores and ensuresefficient energy transfer between the donor and acceptor (or fluorophoreand quencher) when they are on the same side of the membrane, even atlow concentrations. The good energy transfer allows one to separate thedonor emission further from the acceptor absorbance and thus decreasethe spectral crosstalk that contributes to the reduction of thevoltage-sensitive ratio change from theoretical values. Another majoradvantage of a linker is that it converts the system into a unimolecularphenomenon. This greatly simplifies the fluorescence readout, ensures1:1 stoichiometry of donor and acceptor (or fluorophore and quencher),and eliminates the need for optimizing relative loading levels of donorand acceptor for an optimum voltage-sensitive fluorescence change (withthe additional benefit of minimal cellular perturbation and toxicity).The linker group is long enough to span the entire membrane.

The hydrophobic fluorescent molecule and the second reagent are attachedto each other by means of a bifunctional linker. “Linker group” shallmean the “chemical arm” between the first and second reagent. As oneskilled in the art will recognize, to accomplish the requisite chemicalstructure, each of the reactants must contain the necessary reactivegroups.

Representative combinations of such groups are amino with carboxyl toform amide linkages, or carboxy with hydroxy to form ester linkages oramino with alkyl halides to form alkylamino linkages, or thiols withthiols to form disulfides, or thiols with maleimides or alkyl halides toform thioethers. Obviously, hydroxyl, carboxyl, amino and otherfunctionalities, where not present may be introduced by known methods.Likewise, as those skilled in the art will recognize, a wide variety oflinking groups may be employed. The structure of the linkage should be astable covalent linkage formed to attach the two species to each other.The portions of the linker closest to the second reagent may behydrophilic, whereas the portions of the linker closest to the mobilefluorescent molecule should be hydrophobic to permit insertion into themembrane and to avoid retarding the voltage-dependent translocation ofthe anion. The covalent linkages should be stable relative to thesolution conditions under which the cells are loaded. Generallypreferred linking groups will comprise 20–40 bonds from one end to theother and 0–10 heteroatoms (NH, O, S) and may be branched or straightchain. Without limiting the foregoing, it should be evident to oneskilled in the art that only combinations of atoms which are chemicallycompatible comprise the linking group. For example, amide, ester,thioether, thioester, keto, hydroxyl, carboxyl, ether groups in withcarbon-carbon bonds are acceptable examples of chemically compatiblelinking groups. Other chemically compatible linkers may be found in U.S.Pat. Nos. 5,470,997 (col. 2 and col. 4–7) and 5,470,843 (cols. 11–13).

Asymmetric 1,3-substituted thioureas have been prepared for use insynthesizing oxonols with N-substituted linkers containing terminalreactive groups capable of conjugating to appropriate second reagentfluorophores/quenchers. In one example, one of the oxonol substituentsis a pentyl chain (C₅) with a terminal bromide or tosylate group.Thiobarbiturates have been synthesized from these thioureas anddiethylmalonate in ethoxide/ethanol. Mixed pentamethine oxonols preparedfrom 1 equivalent of the barbiturate with functionalized linkers and1,3-dibutyl thiobarbiturate have been characterized. An exemplarysynthesis is depicted in FIG. 11. It will be recognized that oxonolswith alkyl chains of length other than C₅ can be readily prepared bysuch a method and are contemplated as within the scope of thisinvention.

One preferred class of suitable linkers includes bi-functionalizedpolyalkylene glycol oligomers (polyethyleneglycol, polypropyleneglycol,polybutyleneglycol, etc.) of an appropriate length to span the plasmamembrane (25–30 carbon equivalents), for example 8–10 PEG units. Theoxygen (or sulfur, in the corresponding thio-analogs thereof) modulatesthe hydrophobicity and hence translocation rate and loading. Compoundsjoined by such linker groups have the general formulaX—(CH₂)_(m)-Z_(q)-(CH₂)_(m′)-Z′_(q′)-(CH₂)_(m″)-Z″_(q″)-Ywherein:

X is a hydrophobic fluorescent anion;

Y is a fluorescent second reagent;

Z, Z′, Z″ are independently O, S, SS, CO, COO;

m, m′ and m″ are integers from 0 to about 32;

q, q′, and q″ are independently 0 or 1; and

m+q+m′+q′+m″+q″ is from about 20 to 40 (preferably between 25 and 35).

Preferably Z is S, i.e., the linkers are polyalkylene thioethers; m=5,Z=S, q=1, m′=12, Z′=S, q′=1, m″=11, Z″=CO, and q″=1.

Another class of suitable linkers includes functionalized alkyl chainswith terminal thiol groups that form a central disulfide linkage. Thedisulfide functions as a hydrophobic swivel in the center of themembrane. These compounds have the general formulaX—(CH₂)_(n)SS(CH₂)² _(n′)—Ywherein:

X is a hydrophobic fluorescent anion;

Y is a fluorescent second reagent;

n and n′ are integers from 0 to about 32 wherein n+n′ is less than orequal to 33.

As would be readily appreciated by those skilled in the art, the linkergroups may be reacted with appropriately substituted or functionalizedfirst and second fluorophores using conventional coupling chemistries.Further, it is evident that the linker group may be attached to afluorophore at a variety of different positions. Important locations (X)for attachment of the linker in exemplary classes of oxonols arcillustrated in FIG. 12.

VII. Measurement Methods

In one class of embodiments of the present invention, the hydrophobicion fluorescence on one face of the membrane is quenched by a mechanismother than FRET.

FRET has the advantages of working over relatively long distances(compared to the thickness of a typical biological membrane), whichminimizes the necessary concentration of acceptors to give a ratiometricoutput at two emission wavelengths. However, if FRET is too efficientover distances greater than the thickness of the membrane, it can failto provide good discrimination between acceptors on the same vs.opposite sides of the membrane.

The second fluorophore/quencher or luminescent component can be locatedon either the intracellular or the extracellular face, as long as it isspecific to one or the other In the specific examples reported herein,the extracellular surface was targeted for convenience.

FRET or fluorescence quenching is can be detected by emission ratioingthat can distinguish the two populations of the mobile fluorophore,i.e., those bound to the extracellular vs. those bound to theintracellular face of the membrane. In particular, FRET using afluorescent acceptor and a fluorescent donor provides an emission ratiochange that is well suited to laser scanning confocal microscopy andinternally corrects for variations in donor loading, cell thickness andposition (including motion artifacts), and excitation intensity.Emission ratios usually change by larger percentages than eitheremission wavelength signal alone, because the donor and acceptoremissions should change in opposite directions, which reinforce eachother when ratioed. If emission ratioing is not desirable or possible,either wavelength can still be used alone, or the change in donorexcited-state lifetime monitored.

Emission ratios are measured either by changing the passband of awavelength-selective filter in front of a single detector, or preferablyby splitting the emitted light with a dichroic mirror and measuring twowavelength bands simultaneously with two detectors, which may each bepreceded by additional wavelength-selecting filters. In the firstmethod, the wavelength-selective filters may be two or more interferencefilters with different passbands alternately placed in front of thedetector, or they may be a continuously tunable monochromator which isrepeatedly scanned over a wavelength range. The advantage of the firstmethod is that only one detector is used, which economizes on detectorsand avoids the problem of precisely matching two detectors. Theadvantages of the second method, using a dichroic mirror and twoseparate detectors, are that the two emissions may be measured trulysimultaneously rather than sequentially, and that it makes moreefficient use of the photons emitted from the sample.

FRET between a luminescent component and fluorescent component may bepreferred in certain circumstances. Because this approach does notrequire light irradiation of the sample, phototoxicity andautofluorescence of the sample are significantly reduced. Because lightemission is exclusively produced from the luminescent component, theapproach typically exhibits improved specificity and sensitivity.However because the luminescent component typically createssignificantly less light output than is achieved using traditionalfluorescent approaches it is often necessary to use more sensitivedetectors, and to collect and integrate light emission over timecompared to fluorescent emission ratioing.

Another preferred detection method involves the use of time resolvedfluorescence approaches. These methods combine many of the advantages ofthe use of fluorescence, with the enhanced specificity and freedom fromautofluorescence, of luminescence based measurements.

The methods generally encompass the use of a long-lived fluorescentcomponent to provide sustained light emission in the absence ofillumination. Typical components include lanthanides such as terbium oreuropium chelates covalently joined to a polynuclear heterocyclicaromatic sensitizer (for example described in U.S. Pat. No. 5,622,821,issued Apr. 22, 1997) as the first or second reagent. These reagents arepreferred because they are relatively easy to synthesize and attach tomacromolecules, are highly fluorescent, chemically stable and goodresonance energy transfer donors.

The time resolved analysis requires a pulsed excitation source and gateddetectors to enable the transient illumination of the sample and decayof sample autofluorescence. By making fluorescence measurements afterthe decay of autofluorescence, the assay method typically exhibitssignificantly improved signal to noise ratios compared to traditionalfluorescence based approaches. Instrumentation for conducting timeresolved analysis is commercially available from a number of sourcesincluding Hewlett-Packard, LKB, and LJL.

Molecular specificity for particular cell types in a mixed populationmay be further improved by the use of targetable reagents. For example,cell-specific antibodies or lectins as the carriers of the extracellularfluorescent label, or by using naturally fluorescent, or bioluminescentproteins specifically expressed in a given cell type as the intra- orextracellular label. Specifically labeled cells can also reducebackground staining and therefor provide larger fluorescence changes,particularly in complex tissues, where not all the cells may respond toa given stimulus.

High sensitivity is achieved when the voltage sensor (i.e., thehydrophobic anion of the first reagent) translocates at least a fullunit charge nearly all the way through the membrane. Even withoutspecific ion channels or transporters, such translocation can be quiterapid if the ion is negatively charged, delocalized, and hydrophobic.For example, the lipid-soluble non-fluorescent anion of dipicrylamine(2,2′,4,4′,6,6′-hexanitrodiphenylamine) produces displacement currentsin excitable tissue with submillisecond kinetics, comparable in speed tosodium channel gating currents [Benz, R. and Conti, F. 1981. Structureof the squid axon membrane as derived from charge-pulse relaxationstudies in the presence of absorbed lipophilic ions. J. Membrane Biol.59:91–104; Benz, R. and Nonner, W. 1981. Structure of the axolemma offrog myelinated nerve: relaxation experiments with a lipophilic probeion. J. Membrane Biol. 59:127–134; Fernández, J. M., Taylor, R. E., andBezanilla, F. 1983. Induced capacitance in the squid giant axon. J. Gen.Physiol. 82:331–346]. However, voltage sensing should not requirefurther diffusion of the ion through the unstirred aqueous layers,because that slows the response and generates a sustained leakagecurrent.

To create an optical readout from the translocation of the fluorescenthydrophobic ion (i.e., the first reagent) from one side of thebiological membrane to the other side, FRET or fluorescence quenchingbetween the translocating ion and a fluorophore or quencher (i.e., thesecond reagent) fixed to just one face of the membrane is employed. Mostconveniently, the extracellular face is employed.

By way of example, and not limitation, the case where the translocatingions are anionic fluorescent acceptors which absorb at wavelengths thatoverlap with the emission spectrum of the extracellularly fixed donorfluorophores is schematically shown in FIG. 1. At a resting negativemembrane potential (A) permeable oxonols have a high concentration atthe extracellular surface of the plasma membrane and energy transferfrom the extracellularly bound FL-WGA (fluorescein-wheat germagglutinin) is favored. FRET is symbolized by the straight arrow fromlectin to oxonol. At a positive membrane potential (B) the anions arelocated primarily on the intracellular surface of the membrane andenergy transfer is greatly reduced because of their increased meandistance from the donors on the extracellular surface.

The speed of the voltage-sensitive fluorescence response depends on thetranslocation rate of the fluorophore from one site to the other. Thespeed of response for DiSBA-C₆-(3) is shown in FIG. 5 and follows thegeneral equations (1) and (2). As this equation indicates, fluorescentions which jump across the membrane on a millisecond timescale inresponse to biologically significant changes in transmembrane potentialare needed to follow rapid polarization/depolorization kinetics.Slower-jumping ions would not be useful, for example, in following fastelectrical signals in neuronal tissue (a primary application of thecompositions and methods of the present invention). The development anddiscovery of such molecules with the added constraint of beingfluorescent is not trivial.

The mobile hydrophobic anions can be donors rather than acceptors. Eachof the alternatives has its own advantages. An example with thehydrophobic ion being the FRET donor is the DiSBA-C₆-(3)/Texas Red WGAcombination. A primary advantage of this arrangement is that itminimizes the concentration of the hydrophobic dye molecule in themembrane; this reduces toxicity and cellular perturbations resultingfrom the displacement current and any photodynamic effects. Anotheradvantage is the generally higher quantum yields of fluorophores boundin membranes relative to those on proteins or water; this gives betterFRET at a given distance.

Bis-(1,3-dialkyl-2-thiobarbiturate)-trimethineoxonols, where alkyl isn-hexyl and n-decyl (DiSBA-C₆ (3) and DiSBA-C₁₀-(3) respectively) havebeen shown herein to function as donors to Texas Red labeled wheat germagglutinin (TR-WGA) and as acceptors from fluorescein labeled lectin(FL-WGA). In voltage-clamped fibroblasts, the translocation of theseoxonols was measured as a displacement current with a time constant ofabout 2 ms for 100 mV depolarization at 20 C, which equals the speed ofthe fluorescence changes. Fluorescence ratio changes of between 4–34%were observed for a 100 mV depolarization in fibroblasts, astrocytomacells, beating cardiac myocytes, and B104 neuroblastoma cells. The largefluorescence changes allowed high speed confocal imaging.

Single cells were used in the examples so that the optical signals couldbe compared with voltage changes accurately known from traditionalmicroelectrode techniques, such as patch clamping, which are applicableonly to single cells. However, it should be apparent that the dyes canbe used for many applications in which microelectrodes are notapplicable. Comparison with microelectrodes is needed merely foraccurate calibration and proof that the mechanism of fluorescence signalgeneration is as described herein. The two reagent compositions andmethods described herein can either resolve the different electricalpotentials of many neighboring cells or neighboring parts of a singlecell, or give an average reading for all the membrane locations,depending on whether the optical signal is spatially imaged or pooled.

The methods described herein are applicable to a wide variety ofmembranes. In particular, membrane potentials in membranes of isolatedcells, tissues and intact transgenic organisms can be detected andmonitored. The method finds greatest utility with plasma membranes,especially the outermost plasma membrane of mammalian cells.Representative membranes include, but arc not limited to, subcellularorganelles, membranes of the endoplasmic reticulum, secretory granules,mitochondria, microsomes and secretory vesicles. Cell types which can beused include but are not limited to, neurons, cardiac cells, lymphocytes(T and B lymphocytes, nerve cells, muscle cells and the like.

VIII. Drug Screening

The invention also provides methods for screening test samples such aspotential therapeutic drugs that affect membrane potentials inbiological cells. These methods involve measuring membrane potentials asdescribed above in the presence and absence (control measurement) of thetest sample. Control measurements are usually performed with a samplecontaining all components of the test sample except for the putativedrug. Detection of a change in membrane potential in the presence of thetest agent relative to the control indicates that the test agent isactive. Membrane potentials can be also be determined in the presence orabsence of a pharmacologic agent of known activity (i.e., a standardagent) or putative activity (i.e., a test agent). A difference inmembrane potentials as detected by the methods disclosed herein allowsone to compare the activity of the test agent to that of the standardagent. It will be recognized that many combinations and permutations ofdrug screening protocols are known to one of skill in the art and theymay be readily adapted to use with the method of membrane potentialmeasurement disclosed herein to identify compounds which affect membranepotentials. Use of the membrane potential determination techniquedisclosed herein in combination with all such methods are contemplatedby this invention. In a particular application, the invention offers amethod of identifying a compound which modulates activity of an ionchannel, pump, or exchanger in a membrane, comprising:

(a) loading the cells with the first and second reagents, which togethermeasure membrane potential as described above;

(b) determining the membrane potential as described above;

(c) exposing the cells to the test sample;

(d) redetermining the membrane potential and comparing with the resultin (b) to determine the effect of the test sample;

(e) optionally, exposing the membrane to a stimulus which modulates anion channel, pump or exchanger, and redetermining the membrane potentialand comparing with the result in (d) to determine the effect of the testsample on the response to the stimulus.

In another application, the invention offers a method of screening testsamples to identify a compound which modulates the activity of an ionchannel, pump or exchanger in a membrane, comprising:

(a) loading a first set and a second set of cells with first and secondreagents which together measure membrane potential;

(b) optionally, exposing both the first and second set of cells to astimulus which modulates the ion channel, pump or exchanger;

(c) exposing the first set of cells to the test sample;

(d) measuring the membrane potential in the first and second sets ofcells; and

(e) relating the difference in membrane potentials between the first andsecond sets of cells to the ability of a compound in the test sample tomodulate the activity of an ion channel, pump or exchanger in amembrane.

In another aspect the method includes the use of a membrane potentialmodulator to set the resting, or stimulated membrane potential to apredefined value. At this predefined value the cell is unable to rapidlyreset the membrane potential in response to the transient activation ofthe target ion channel. Thus as a result of the artificial membranepotential, even transient activation of the target ion channel leads toa prolonged, measurable change in membrane potential that is typicallylarger and more conveniently measured during screening. This methodenables the detection of very transient ion channel currents, which arenormally difficult to measure in a convenient format. Such channelsinclude rapidly inactivating sodium channels, T-type calcium channelsand ligand-gated channels.

In another aspect of this method, the membrane potential modulatorcomprises a ligand dependent ion channel. Activation of the ligand gatedion channel causes a voltage change in the cell that provides thestimulus to activate a voltage-dependent channel target. The ligand canbe added directly or released via UV flash uncaging to enable rapidlyinactivating voltage dependent ion channels to be monitored.

This approach enables voltage activated ion channels to be activatedwithout using traditional electrophysiological (i.e. voltage clamp orpatch clamp) methods, that are not readily amendable to high throughputscreening.

Ion channels of interest include, but are not limited to, sodium,calcium, potassium, nonspecific cation, and chloride ion channels, eachof which may be constitutively open, voltage-gated, ligand gated, orcontrolled by intracellular signaling pathways.

Biological cells which can be screened include, but are not limited toprimary cultures of mammalian cells, transgenic organisms and mammaliantissue. Cells in screening assays may be dissociated either immediatelyor after primary culture. Cell types include, but are not limited towhite blood cells (e.g. leukocytes), hepatocytes, pancreatic beta-cells,neurons, smooth muscle cells, intestinal epithelial cells, cardiacmyocytes, glial cells, and the like. The invention also includes the useof recombinant cells into which ion transporters, ion channels, pumpsand exchangers have been inserted and expressed by genetic engineering.Many cDNA sequences for such transporters have been cloned (see U.S.Pat. No. 5,380,836 for a cloned sodium channel) and methods for theirexpression in cell lines of interest is within the knowledge of one ofskill in the art (see, U.S. Pat. No. 5,436,128). Representative culturedcell lines derived from humans and other mammals include LM (TK−) cells,HEK293 (human embryonic kidney cells), 3T3 fibroblasts, COS cells, CHOcells, RAT1 and HLHepG2 cells.

The screening methods described herein can be made on cells growing inor deposited on solid surfaces. A common technique is to use amicrotiter plate well wherein the fluorescence measurements are made bycommercially available fluorescent plate readers. The invention includeshigh throughput screening in both automated and semiautomated systems,such as described in PCT publication No. WO 98/52047, and U.S. patentapplication Ser. No. 09/122,544 filed Jul. 24, 1999 entitled “Detectorand Screening Device for Ion Channels.” One such method is to use cellsin Costar 96 well microtiter plates (flat with a clear bottom) andmeasure fluorescent signal with CytoFluor multiwell plate reader(Perseptive Biosystems, Inc., MA) using two emission wavelengths torecord fluorescent emission ratios.

The invention may be better understood with reference to theaccompanying examples, which are intended for purposes of illustrationonly and should not be construed as in any sense limiting the scope ofthe invention as defined in the claims appended hereto.

EXAMPLES Example I—Synthesis of Oxonol Dyes

All starting materials and reagents were of the highest purity available(Aldrich; Milwaukee, Wis.) and used without further purification, exceptwhere noted. Solvents were HPLC grade (Fisher) and were dried overactivated molecular sieves 3 Å. NMR spectra were acquired on a VarianGemini 200 MHz spectrometer (Palo Alto, Calif.). The spectra werereferenced relative to the residual solvent peak (CHCl₃, =7.24 ppm).Fluorescence spectra were taken on a SPEX Fluorolog-2 (Edison, N.J.) andwere corrected for lamp and detector wavelength variations using themanufacturer supplied correction files.

Bis-(1,3-dibutyl-2-thiobarbiturate)-trimethineoxonol DiSBA-C₄-(3):

DiSBA-C₄-(3) was synthesized based on the procedure for the ethylderivative [British Patent 1,231,884]. 1,3-di-butyl-thiobarbiturate (500mg, 2 mmol) was dissolved in 700 μL of pyridine. To this solution, amixture of 181 μL (1.1 mmol) of malonaldehyde bis(dimethyl acetal) and100 μL of 1 M HCl was added. The solution immediately turned red. After3 h, half of the reaction mixture was removed and 2 equiv. of theprotected malonaldehyde was added every hour for 4 h to the remainingmixture. The reaction mixture was then filtered to collect purple/blackcrystals of the DiSBA-C₄-(3) pyridinium salt. After washing the crystalswith water and then drying under vacuum (0.5 torr), 67.2 mg of pureproduct was collected. ¹H NMR (CDCl₃): 8.91 (2H, d, J=5.1 Hz, py), 8.76(1H, t, J=13.7 Hz, central methine), 8.52 (1H, t, J=8.0 Hz, py), 8.16(2H, d, J=13.9 Hz, methine), 8.00 (2H, dd, J₁˜J₂=6.9 Hz, py), 4.47 (8H,cm, NCH ₂CH₂CH₂CH₃), 1.69 (8H, cm, NCH₂CH ₂CH₂CH₃), 1.39 (8H, cm,NCH₂CH₂CH ₂CH₃), 0.95 (12H, t, J=6.4 Hz, methyl).

To prepare 1,3-di-butyl-thiobarbiturate, 1.22 g of Na (53 mmol) wasslowly dissolved in 20 mL of dry ethanol under argon. To the ethoxidesolution, 8.5 g (8 mL, 53 mmol) of diethyl malonate followed by 5 g(26.5 mmol) of dibutyithiourea were added. The reaction mixture washeated and refluxed for 3 days. After cooling, the mixture was filtered.The filtrate was clarified with addition of water. Concentrated HCl wasthen added until the pH was 1–2. The acidic filtrate was then extracted3× with hexanes. The extract was concentrated and 5.5 g of crude productprecipitated out of solution. The solid was recrystallized from methanolwith addition of small amounts of water yielding 4.23 g of the purebarbituric acid (65%). ¹H NMR (CDCl₃): 4.33 (4H, cm, NCH ₂CH₂CH₂CH₃),3.71 (2H, s, ring CH₂), 1.63 (4H, cm, NCH₂CH ₂CH₂CH₃), 1.35 (4H, cm,NCH₂CH₂CH ₂CH₃), 0.94 (6H, t, J=6.2 Hz, methyl).

Bis-(1,3-dihexyl-2-thiobarbiturate)-pentamethineoxonol (DiSBA-C₆-(5)):1,3-dihexyl-2-thiobarbituric acid (200 mg, 0.64 mmol) andglutacondialdehyde dianil monohydrochloride (whose Chem. Abs. name isN-[5-(phenylamino)-2,4-pentadienylidene]benzenamine, monohydrochloride)(91 mg, 0.32 mmol) were mixed in 1 mL pyridine. Within 10 s, thesolution turned blue. After letting the reaction mixture stir for 1.5 h,the solvent was removed under high vacuum. The residue was dissolved inCHCl₃ and chromatographed on silica gel eluting with a (93:7) CHCl₃/MeOHsolution. The pure blue oxonol (72 mg) was recovered. ¹HNMR(CDCl₃/CD₃OD): d 7.60–7.80 (cm, 4H, methines), 7.35 (t, J=11.3 Hz, 1H,central methine), 4.31 (cm, 8H, NCH₂R), 1.57 (cm, 8H, NCH₂CH₂R), 1.20(br m, 24H, bulk methylenes), 0.74 (br t, 12H, methyl).

Other oxonols were made using the same procedure, starting with theappropriate thiourea prepared from requisite primary amine and carbondisulfide [Bortnick, N., Luskin, L. S., Hurwitz, M. D., and Rytina, A.W. 1956. t-Carbinamines, RR′R″CNH₂. III. The preparation of isocyanates,isothiocyanates and related compounds. J. Am. Chem. Soc. 78:4358–4361].An exemplary synthesis of Di-SBA-C₆-(3) is depicted in FIG. 13.

Example II—Synthesis of Fluorescent Phospholipids

Cou-PE:3-amidoglycine-6-chloro-7-butyryloxy coumarin was synthesized asdescribed in pending U.S. patent application, Ser. No. 08/407,554, filedMar. 20, 1995 as set out below. For synthesis of 2,4dihydroxy-5-chlorobenzaldehyde, 21.7 g (0.15 Mol) 4-chlororesorcinolwere dissolved in 150 ml dry diethyl ether and 27 g finely powdered zinc(II) cyanide and 0.5 g potassium chloride were added with stirring. Thesuspension was cooled on ice. A strong stream of hydrogen chloride gaswas blown into the solution with vigorous stirring. After approximately30 minutes the reactants were dissolved. The addition of hydrogenchloride gas was continued until it stopped being absorbed in the ethersolution (approx 1 hour). During this time a precipitate formed. Thesuspension was stirred for one additional hour on ice. Then the solidwas let to settle. The ethereal solution was poured from the solid. Thesolid was treated with 100 g of ice and heated to 100 degrees C. in awater bath. Upon cooling the product crystallized in shiny plates fromthe solution. They were removed by filtration on dried over potassiumhydroxide. The yield was 15.9 g (0.092 Mol, 61%). ¹H NMR (CDCl₃): 6.23ppm (s, 1H, phenol), 6.62 ppm (s, 1H, phenyl), 7.52 ppm (s, 1H, phenyl),9.69 ppm (s, 1H, formyl), 11.25 ppm (s, 1H, phenol).

To prepare 3-carboxy 6-chloro 7-hydroxy coumarin, 5.76 g (0.033 Mol)2,4-dihydroxy-5-chlorobenzaldehyde and 7.2 g (0.069 Mol) malonic acidwere dissolved in 5 ml warm pyridine. 75 microliters aniline werestirred into the solution and the reaction let to stand at roomtemperature for 3 days. The yellow solid that formed was broken intosmaller pieces and 50 ml ethanol was added. The creamy suspension wasfiltered through a glass frit and the solid was washed three times with1 N hydrochloric acid and then with water. Then the solid was stirredwith 100 ml ethyl acetate, 150 ml ethanol and 10 ml half concentratedhydrochloric acid. The solvent volume was reduced in vacuo and theprecipitate recovered by filtration, washed with diethyl ether and driedover phosphorous pentoxide. 4.97 g (0.021 Mol, 63%) of product wasobtained as a white powder. ¹H NMR (dDMSO): 6.95 ppm (s, 1H), 8.02 ppm(s, 1H), 8.67 ppm (s, 1H).

To prepare 7-butyryloxy-3-carboxy-6-chlorocoumarin, 3.1 g (12.9 mMol)3-carboxy-6-chloro-7-hydroxycoumarin were dissolved in 100 ml dioxaneand treated with 5 ml butyric anhydride, 8 ml pyridine and 20 mgdimethyl aminopyridine at room temperature for two hours. The reactionsolution was added with stirring to 300 ml heptane upon which a whiteprecipitate formed. It was recovered by filtration and dissolved in 150ml ethyl acetate. Undissolved material was removed by filtration and thefiltrate extracted twice with 50 ml 1 N hydrochloric acid/brine,(1:1)and then brine. The solution was dried over anhydrous sodium sulfate.Evaporation in vacuo yielded 2.63 g (8.47 mMol, 66%) of product. ¹H NMR(CDCl₃): 1.08 ppm (t, 3H, J=7.4 Hz, butyric methyl), 1.85 ppm (m, 2H, J₁J₂=7.4 Hz, butyric methylene), 2.68 ppm (t, 2H, J=7.4 Hz, butyricmethylene), 7.37 ppm (s, 1H, coumarin), 7.84 ppm (s, 1H, coumarin), 8.86ppm (s, 1H, coumarin).

Preparation of7-butyryloxy-3-benzyloxycarbonylmethylaminocarbonyl-6-chlorocoumarin iseffected as follows. 2.5 g (8.06 mMol)7-Butyryloxy-3-carboxy-6-chlorocoumarin, 2.36 g hydroxybenztriazolehydrate (16 mMol) and 1.67 g (8.1 mMol) dicyclohexyl carbodiimide weredissolved in 30 ml dioxane. A toluene solution of O-benzylglycine[prepared by extraction of 3.4 g (10 mMol) benzylglycine tosyl salt withethyl acetate-toluene-saturated aqueous bicarbonate-water (1:1:1:1, 250ml), drying of the organic phase with anhydrous sodium sulfate andreduction of the solvent volume to 5 ml] was added dropwise to thecoumarin solution. The reaction was kept at room temperature for 20hours after which the precipitate was removed by filtration and washedextensively with ethylacetate and acetone. The combined solventfractions were reduced to 50 ml on the rotatory evaporator upon whichone volume of toluene was added and the volume further reduced to 30 ml.The precipitating product was recovered by filtration and dissolved in200 ml chloroform-absolute ethanol (1:1). The solution was reduced to 50ml on the rotatory evaporator and the product filtered off and dried invacuo yielding 1.29 g of the title product. Further reduction of thesolvent volume yielded a second crop (0.64 g). Total yield: 1.93 g (4.22mMol, 52%). ¹H NMR (CDCl₃): 1.08 ppm (t, 3H, J=7.4 Hz, butyric methyl),1.84 ppm (m, 2H, J₁ J₂=7.4 Hz, butyric methylene), 2.66 ppm (t, 2H,J=7.4 Hz, butyric methylene), 4.29 ppm (d, 2H, J=5.5 Hz, glycinemethylene), 5.24 ppm (s, 2H, benzyl), 7.36 ppm (s, 1H, coumarin), 7.38ppm (s, 5H, phenyl), 7.77 ppm (s, 1H, coumarin), 8.83 ppm (s, 1H,coumarin), 9.15 ppm (t, 1H, J=5.5 Hz, amide).

7-Butyryloxy-3-carboxymethylaminocarbonyl-6-chlorocoumarin was preparedas follows. 920 mg (2 mMol)7-butyryloxy-3-benzyloxycarbonylmethylaminocarbonyl-6-chlorocoumarinwere dissolved in 50 ml dioxane. 100 mg palladium on carbon (10%) and100 microliters acetic acid were added to the solution and thesuspension stirred vigorously in a hydrogen atmosphere at ambientpressure. After the uptake of hydrogen seized the suspension wasfiltered. The product containing carbon was extracted five times with 25ml boiling dioxane. The combined dioxane solutions were let to cool uponwhich the product precipitated as a white powder. Reduction of thesolvent to 20 ml precipitates more product. The remaining dioxanesolution is heated to boiling and heptane is added until the solutionbecomes cloudy. The weights of the dried powders were 245 mg, 389 mg and58 mg, totaling 692 mg (1.88 mMol, 94%) of white product.

¹H NMR (dDMSO): 1.02 ppm (t, 3H, J=7.4 Hz, butyric methyl), 1.73 ppm (m,2H, J₁ J₂=7.3 Hz, butyric methylene), 2.70 ppm (t, 2H, J=7.2 Hz, butyricmethylene), 4.07 ppm (d, 2H, J=5.6 Hz, glycine methylene), 7.67 ppm (s,1H, coumarin), 8.35 ppm (s, 1H, coumarin), 8.90 ppm (s, 1H, coumarin),9.00 ppm (t, 1H, J=5.6 Hz, amide).

6-chloro-7-(n-butyryloxy)coumarin-3-carboxamidoacetic acid (26.2 mg, 100mmol) was dissolved in 2 mL of 1:1 CHCl₃/dioxane. Isobutylchloroformate(14.3 mL, 110 mmol) was added under Argon at 4° C. and left stirring for30 min. Separately, dimyristoylphosphatidylethanolamine (DMPE) (20 mg,31.5 mmol) was dissolved in 1 mL of CHCl₃ with 1 drop of dry MeOH and 6mL (34.5 mmol) of diisopropylethylamine (DIEA) added. The mixedanhydride solution was then pipetted into the phospholipid solution.After 2 h, the solvent was removed under vacuum. The residue wasdissolved in 3 mL of MeOH and mixed with 3 mL of 0.25 M NaHCO₃. Thesolution almost immediately turn yellow and was stirred for 15 min. Thesolution was then extracted 3–5 times with CHCl₃. A bad emulsion isformed. The extracts were combined and concentrated. The residue wasdissolved in 1 mL 1:1 MeOH/H₂O and purified on a C₁₈ reverse phasecolumn (1.7×7 cm). Eluting with the same solvent, a fluorescent bandpassed through the column, followed by a slower one. The solventpolarity was decreased to 9:1 MeOH/H₂O and the major yellow band theneluted off the column. After concentration and drying 2.5 mg (2.74 mmol)of pure product was collected.

¹HNMR (CD₃OD): d 8.72 (s, 1H, coumarin), 7.81 (s, 1 H, coumarin), 6.84(s, 1H, coumarin), 5.25 (cm, 2H), 4.43 (dd, J₁=12.1 Hz, J₂=3.2 Hz), 4.22(d, J=6.6 Hz, 1 H), 4.13 (s, ˜4H), 3.7–4.1 (cm, ˜11H), 3.47 (cm, ˜3H),3.2–3.3 (˜q), 2.31 (cm ˜7H), 1.57 (br s, ˜8H, CH₂ a to carbonyl),1.2–1.5 (cm, ˜63H, bulk CH₂'s), 0.92 (unres t, ˜12H, CH₃). Electrospray(neg. ion) MS [MeOH/H₂O: 95/5] (peak, rel. int.) 456.8 (M⁻², 20), 524.5(50), 704.9 (6), 734.7 (100), 913.9 (M⁻¹, 95); deconvoluted M=915.3 amu;calc. M=915.5 amu. UV-vis (MeOH/HBSS; 2/1) l_(max)=414 nm. Fluorescence(MeOH/HBSS; 2/1) l_(emax)=450 nm, Quantum Yield=1.0.

Cy5-PE:DMPE (1.0 mg, 1.6 mmol) was dissolved in 650 mL of (12:1)CHCl₃/MeOH and DIEA (1 mL, 5.7 mmol) was added. Separately, Cy5-OSu(Amersham; Arlington Heights, Ill.), the N-hydroxysuccinimide ester ofN-ethyl-N′-(5-carboxypentyl)-5,5′-disulfoindodicarbocyanine, (0.8 mg, 1mmol) was dissolved in 150 mL of (2:1) CHCl₃/MeOH and added to thephospholipid solution. After 3 h, the solvent was removed under vacuum.The residue was dissolved in MeOH and loaded on a C₁₈ reverse phasecolumn (1×10 cm) equilibrated with 1:1 MeOH/H₂O. Eluting with the samesolvent, the hydrolyzed ester was removed. The polarity was decreased to9:1 MeOH/H₂O and the pure blue product was eluted off the column,yielding 400 mg (310 nmol, 31%).

Example III—Synthesis of Linker for Donors and Acceptors

This example is with reference to FIGS. 14–16.

12-p-methoxybenzylthio-1-dodecanol (1): Na (800 mg, 34.8 mmol) wasdissolved in 30 mL of dry MeOH. Under argon, p-methoxybenzylmercaptan(2.75 mL, 3.04 g, 19.7 mmol) was added to the methoxide solution. Aftera few minutes, 12-bromododecanol (2.5 g, 9.43 mmol) was dropped into thereaction mixture. Within 5 minutes a solid began to come out ofsolution. After a minimum of 3 h, the reaction was filtered and washed3× with cold MeOH, yielding 2.874 g (8.49 mmol, 90%) of pure productafter drying. ¹H NMR (CDCl₃): d 7.23 (d, J=8.8 Hz, 2H, AA′ of AA′ BB′aromatic system), 6.85 (d, J=8.8 Hz, 2H, BB′ of AA′ BB′ aromaticsystem), 3.80 (s, 3H, methoxy), 3.66 (s, 2H, benzyl), 3.64 (dt, J₁=6.6Hz, J₂=5.5 Hz, 2H, RCH₂OH), 2.40 (t, J=7.3 Hz, 2H, RSCH₂R), 1.50–1.65(cm, 4H, CH₂ b to heteroatoms), 1.2–1.4 (cm, 16H, bulk methylenes).

12-p-methoxybenzylthio-1-bromododecane (2): (1) (500 mg, 1.48 mmol) wasmixed with carbon tetrabromide (611 mg, 1.85 mmol) in 2.5 mL CH₂Cl₂ andcooled in an ice bath until solid began to come out of solution. The icebath was removed and triphenylphosphine (348 mg, 2.22 mmol) was added tothe reaction. The solution immediately turned yellowish. The startingmaterial had been consumed after 30 min according to TLC (EtOAc/Hex,1:1). The solvent was removed and 50 mL of hexane was added to the solidresidue. After stirring overnight, the solution was filtered andconcentrated to a solid. The solid was then mixed with about 10–15 mL ofhexane and again filtered. The concentrated filtrate yielded 537 mg(1.34 mmol, 91%) of pure product after drying. ¹H NMR (CDCl₃): d 7.23(d, J=8.6 Hz, 2H, AA′ of AA′ BB′ aromatic system), 6.85 (d, J=8.7 Hz,2H, BB′ of AA′ BB′ aromatic system), 3.80 (s, 3H, methoxy), 3.67 (s, 2H,benzyl), 3.41 (t, J=6.9 Hz, 2H, RCH₂Br), 2.40 (t, J=7.3 Hz, 2H, RSCH₂R),1.86 (cm, 2H, CH₂ b to Br), 1.15–1.45 (cm, 18H, bulk methylenes).

12-(12-p-methoxybenzylthio-1-dodecylthio)-dodecanoic acid (3): Na (116mg, 5 mmol) was dissolved it dry MeOH. 12-mercapto-1-dodecanoic acid(340 mg, 1.46 mmol)—synthesized according to JACS 115, 3458–3474,1993—was added to the methoxide solution. After stirring with someheating for 5 min, (2) (497 mg, 1.24 mmol) was added to the reaction.The reaction became very viscous and an additional 1.75 mL of MeOH wasintroduced. The reaction was then left overnight. The reaction wasquenched with 10% acetic acid. The paste-like reaction mixture wastransferred to a 500 mL separatory funnel dissolved in equal volumes ofEtOAc/Hex (1:1) and the acetic acid solution. The organic layer wasseparated. The aqueous layer was then extracted two more times. Thecombine extracts were concentrated yielding 740.3 mg (1.34 mmol) ofcrude product. The excess acetic acid was removed as a tolueneazeotrope. The solid was crystallized from isopropyl ether giving 483 mg(71%). TLC and NMR show an impurity believed to be a disulfide sideproduct. The material was further purified by flash chromatographyeluting with CHCl₃/MeOH/AA (99:0.5:0.5) yielding 334 mg (0.604 mmol,49%) of pure product. ¹H NMR (CDCl₃): d 9.45 (brs, 1H, COOH), 7.23 (d,J=8.8 Hz, 2H, AA′ of AA′ BB′ aromatic system), 6.85 (d, J=8.7 Hz, 2H,BB′ of AA′BB′ aromatic system), 3.80 (s, 3H, methoxy), 3.66 (s, 2H,benzyl), 2.50 (t, J=7.3 Hz, 4H, RCH₂SCH₂R), 2.40 (t, J=7.3 Hz, 2H,RSCH₂R), 2.35 (t, J=7.5 Hz, 2H, RCH₂COOH), 1.5–1.7 (cm, 8H, CH₂ b toheteroatoms), 1.15–1.45 (cm, 30H, bulk methylenes). ¹³C NMR (CDCl₃): d179.5 (COOH), 129.9 (aromatic, 2C), 113.9 (aromatic, 2C), 55.2 (MeOR),35.6 (CH₂), 33.7 (CH₂), 32.2 (CH₂), 31.3 (CH₂), 29.7 (CH₂), 29.4 (CH₂),29.2 (CH₂), 28.9 (CH₂), 24.6 (CH₂).

1-butyl-3-(12-(12-pentylthio-1-dodecylthio)-dodecanoic acid)thiobarbiturate (13): (3) (73.8 mg, 133.5 μmol) was deprotected in 2 mLof dry TFA/anisole (14:1) at 70° C. for 2.5 h. The solvent was removedunder vacuum and the residue was dissolved in 10 mL dry EtOH. Sodiumborohydride (330 mg) was added and the mixture was stirred overnight.The solution was then acidified with conc. HCL until gas stoppedevolving. The solution was then extracted with ether 4×. The combinedextracts were concentrated leaving a white solid. The solid was thendissolved and concentrated 2× with degassed MeOH. After drying on thehigh vacuum, 69.5 mg of solid was recovered. It was estimated by TLCthat this solid was 1:1 the deprotected product anddi-p-methoxyphenylmethane, (104 μmol, 78%). The deprotected linker wasdissolved in 0.5 mL dry DMF, with heating. NaH (˜550 μmol) was addedwhich caused some gas evolution. (8) (38.5 mg, ˜100 μmol) was then addedto the reaction in 100 uL DMF and the reaction was left overnight at 60°C. TLC in EtOAc/MeOH/AA (90:8:2) indicated that a new more non-polarbarbituric acid had been formed. The solvent was removed and the residuewas dissolved in EtOAc/Hex (1:1) and washed with water. The material wasthen purified by chromatography, eluting with EtOAc/MeOH/AA (90:8:2).NMR of the product showed resonance from barbituric acid and the linker.Electrospray (neg. ion) MS [MeOH/H₂O: 95/5] (peak, rel. int.) 516.4(95), 699.4 (M⁻¹, 100), 715.1 (M⁻¹+16, 40), 1024.0 (M⁻¹+32, 25); calc.M⁻¹=700.1 amu. The ether linkers appears to be partially oxidized tosulfoxides.

1-(1,3-dibutylthiobarbiturate)-3-(1-butyl-3-(12-(12-pentylthio-1-dodecylthio)-dodecanoicacid) thiobarbiturate) trimethineoxonol (14): (3) (73.8 mg, 133.5 mol)was deprotected in 2 mL of dry TFA/anisole (14:1) at 7° C. for 2.5 h.The solvent was removed under vacuum and the residue was dissolved in 20mL dry EtOH. Sodium borohydride (330 mg) was added and the mixture wasstirred overnight. The solution was then acidified with conc. HCL untilgas stopped evolving. The solution was then extracted with ether 4×. Thecombined extracts were concentrated leaving a white solid. The solid wasthen dissolved and concentrated 2× with degassed MeOH. After drying onthe high vacuum, 71.1 mg of solid was recovered. It was estimated by TLCthat this solid was 1:1 the deprotected product anddi-p-methoxyphenylmethane, (106 μmol, 79%). The deprotected linker wasdissolved in 1 mL dry DMF, with heating. NaH (˜350 μmol) was added whichcaused some gas evolution. (12) (35.5 μmol) was then added to thereaction in 200 μL DMF. The reaction did not seem to be proceeding after1 h, so 4 mg of N.H. (60%) was added to the reaction mixture. Thesolution now appeared orange instead of red and a second non-polaroxonol began to form. The reaction, heated at 60° C., was allowed to gofor 18 h. Half of the reaction mixture was worked up as follows. Thereaction mixture was transferred to a 30 mL separatory funnel in ˜12 mLtoluene. About 4 mL a 10% acetic acid solution and 3 mL of water wereadded. Most of the oxonol partitioned into the organic layer, which waswashed 3× with acetic acid solution/water (1:1). The organic layer wasthen concentrated and purified by flash chromatography (2.5×18 cm). Thecolumn was packed and first eluted with CHCl₃/MeOH/AA (93:5:2). After 1non-polar oxonol was removed, the solvent polarity was increased toCHCl₃/MeOH/AA (90:8:2). This caused the oxonol product to elute off thecolumn. After concentrating the fractions and drying, 7.2 mg pureproduct (7.25 μmol, 20%) was attained. ¹H NMR (CDCl₃/MeOH): d 8.54 (t,J=13.8 Hz, 1H, central methine), 7.97 (d, J=14.2 Hz, 2H, methines), 4.39(cm, 8H, NCH₂R), 2.46 (t, J=7.3 Hz, 8H, RCH₂SCH₂R), 2.2 (t, 2H,RCH₂COOH), 1.5–18 (bulk methylenes), 1.2–1.4 (bulk methylenes), 0.92 (t,J=7.2 Hz, 9H, methyls). Electrospray (neg. ion) MS [MeOH/H₂O: 95/5](peak, rel. int.) 683 (50), 977.8 (30), 992.1 (M⁻¹, 100), 1008.1(M⁻¹+16, 40), 1024.0 (M⁻¹+32, 10); calc. M⁻¹=992.5 amu. The +16 and +32peaks suggest oxidation of thioethers to sulfoxide groups. (14) has alsobeen successfully synthesized from (13) using (10) in a similar fashionas that described in the synthesis of (11).

1-(1,3-dibutylthiobarbiturate)-3-(1-butyl-3-(12-(12-pentylthio-1-dodecylthio)-N-hydroxysuccinimidedodecanoate) thiobarbiturate) trimethineoxonol (15): 22.5 μmol of (14)was reacted with disuccinimidyl carbonate (57 mg, 225 μmol) in 0.5 mLCH₂Cl₂ in the presence of DIEA (39 μL, 225 mmol). After 1.5 hours, TLC(EtOAc/MeOH) (9:1) indicated that 3 new non-polar bands had been formed.The solvent was removed and two non-polar oxonol bands were purified byflash chromatography, eluting with (EtOAc/MeOH) (95:5). Electrospray(neg. ion) MS [MeOH/H₂O: 95/5] (peak, rel. int.) 1089.3 (M⁻¹, 20),1105.1 (M⁻¹+16, 100), 1121.0 (M⁻¹+32, 60); calc. M⁻¹=1089.5 amu.

Example IV—Measurement of Membrane Potential with Oxonol Dyes as FRETAcceptors and Fluorescent Lectins as FRET Donors

FL-WGA was purchased from Sigma Chemical Co. (St. Louis, Mo.). TR-WGAwas prepared from WGA and Texas Red (Molecular Probes; Eugene, Oreg.) ina 100 mM bicine buffer at pH 8.5. A 73 μM solution of WGA was reactedwith a 6-fold excess of Texas Red for 1 h at room temperature. Theprotein conjugate was purified on a G-25 Sephadex column.

All cells were grown and handled like L-M(TK⁻) except where noted.L-M(TK⁻) cells were grown in Dulbecco's Modified Eagle Media (Gibco;Grand Island, N.Y.) with 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin (PS) (Gemini; Calabasas, Calif.). B104 cells weredifferentiated with 1 μM retinoic acid for 5 days prior to use. Thecells were plated on glass coverslips at least one day before use. Theadherent cells were washed and maintained in 2.5–3.0 mL of HBSS with 1g/L glucose and 20 mM HEPES at pH 7.4. A freshly prepared 75 μM aqueoussolution of the appropriate oxonol was made prior to an experiment froma DMSO stock solution. The cells were stained by mixing 100 μL of theoxonol solution with 750 μL of the bath and then adding the dilutedsolution to the cells. The dye was left for 30–40 minutes at a bathconcentration of 2.3 M. 1.5 mM β-cyclodextrin in the bath solution wasnecessary for cell loading of DiSBA-C₆-(3). The butyl and ethylderivatives were water-soluble enough to load cells with outβ-cyclodextrin complexation. DiSBA-C₁₀-(3) was loaded in a pH 7.4solution containing 290 mM sucrose and 10 mM HEPES, 364 mOsm, for 10 minat a bath concentration of 10 M. DiSBA-C₁₀-(3) labeling was quenched byreplacing the bath with HBSS solution. The cells were stained with 15g/mL of FL-WGA for 15 minutes. The B104 cells required a 125 g/mL bathconcentration to give satisfactory lectin staining. The excess dyes wereremoved with repeated washes with HBSS. If the excess ethyl or butyloxonol derivatives were left in the bath, slow currents and fluorescencechanges due to redistribution of the dyes into the cell were observedduring depolarizations greater than 1 s. The cardiac myocytes[Henderson, S. A., Spencer, M., Sen, A., Kumar, C., Siddiqui, M. A. Q.,and Chien, K. R. 1989. Structure organization, and expression of the ratcardiac myosin light chain-2 gene. J. Biol. Chem. 264:18142–18146] werea gift of Professor Kenneth Chien, UCSD. The Jurkat lymphocytesuspensions were grown in RPMI media with 5% heat inactivated FBS and 1%PS. 15–20 mL aliquots of the cellular suspension were washed three timesbefore and after dye staining by centrifugation at 100×g for 4 minutesfollowed by additions of fresh HBSS.

The fluorescently labeled cells were excited with light from a 75 Wxenon lamp passed through 450–490 nm excitation interference filters.The light was reflected onto the sample using a 505 nm dichroic. Theemitted light was collected with a 63× Zeiss (1.25 or 1.4 numericalaperture) lens, passed through a 505 nm long pass filter and directed toa G-1B 550 nm dichroic (Omega; Brattleboro, Vt.). The reflected lightfrom this second dichroic was passed through a 515 DF35 bandpass filterand made up the FL-WGA signal. The transmitted light was passed througha 560 or 570 LP filter and comprised the oxonol signal. For experimentsusing the oxonol as a donor to TR-WGA, the 550 nm dichroic was used forexcitation and a 580 nm dichroic was used to split the emission. Thelong wavelength Texas Red fluorescence was passed through a 605 nm DF55bandpass filter. Voltage dependent fluorescence changes in single cellswere measured using a Nikon microscope attached to a Photoscan IIphotometer equipped with two R928 PMTs for dual emission recordings. A7-point Savitsky-Golay smoothing routine was applied to all optical data[Savitsky, A. and Golay, M. J. E. 1964. Smoothing and differentiation ofdata by simplified least squares procedure. Anal. Chem. 36:1627–1639],unless otherwise noted. The 1–2 KHz single wavelength data was acquiredwith an Axobasic program that used the TTL pulse counting routine LEVOKEConfocal images were acquired using a home built high speed confocalmicroscope [Tsien, R. Y. and B. J. Bacskai. 1994. Video-rate confocalmicroscopy. In Handbook of Biological Confocal Microscopy. J. B. Pawley,editor. Plenum Press, New York]. The cell was voltage-clamped at aholding potential of −70 mV. After a 200 ms delay, the cell was given a200 ms depolarizing square voltage pulse to 50 mV. Pseudocolor imagesshowing the ratio of the Fl-WGA to oxonol emissions were collected every67 ms and clearly showed a change in ratio, localized to the plasmamembrane, upon depolarization of the cell to +50 mV.

Patch clamp recording were made using an Axopatch 1-D amplifier equippedwith a CV-4 headstage from Axon Instruments (Foster City, Calif.). Thedata were digitized and stored using the PCLAMP software. The pH 7.4intracellular solution used contained 125 mM potassium gluconate, 1 mMCaCl₂.2H₂O, 2 mM MgCl₂.6H₂O, 11 mM EGTA, and 10 mM HEPES. For the B104cells, 4 mM ATP and 0.5 mM GTP were added.

The quantum yield of DiSBA-C₆-(3) was determined relative to rhodamine Bin ethanol (_(F)=0.97) [Weber, G. and Teale, F. W. K. 1957.Determination of the absolute quantum yield of fluorescent solutions.Faraday Soc. Trans. 53:646–655]. R_(o) was calculated following standardprocedures [Wu, P. and Brand, L. 1994. Resonance energy transfer:methods and applications. Anal. Biochem. 218:1–13]. The spectra ofFL-WGA in HBSS and DiSBA-C₆-(3) in octanol were used to determine theoverlap integral. Values of 1.4 and 0.67 were used for the index ofrefraction and orientation factor respectively.

Symmetrical bis(thiobarbiturate)oxonols were chosen as likely candidatesfor rapidly translocating fluorescent ions based on the above designcriteria. The strong absorbance maximum (˜200,000 M⁻¹cm⁻¹) at 540 nm andgood quantum yield (0.40) in membranes makes them desirable for use as afluorescence donors or acceptors in cells. The fluorescence excitationand emission spectra of DiSBA-C₆-(3) is shown in FIG. 2 along with thosefor FL-WGA and TR-WGA. The excitation spectra are the shorter of eachpair. Octanol was selected as the oxonol solvent in order to mimic themembrane environment.

The translocation rates were studied in L-M(TK⁻) cells using whole-cellvoltage clamp recording. The L-M(TK⁻) cells were chosen because theyhave very low background currents and are easy to patch clamp. Thesecells have a resting potential of −5 mV and no evident voltage activatedcurrents.

Displacement currents from DiSBA-C₆-(3) at 20 C are displayed in FIG. 3.12.5 ms long voltage steps at 15 mV increments were applied to the cell,from a holding potential of −70 mV. The larger, faster transients due tosimple membrane capacitance transient could be minimized using thecapacitance and series resistance compensation capabilities of theAxopatch amplifier, allowing the displacement currents to be clearlyobserved. The currents are due to redistribution of the membrane-boundoxonol in response to 8 depolarizations. The time constant for thedisplacement current is 2 ms for 120 mV depolarization. Equal amounts ofcharge move at the onset and conclusion of the voltage step, but inopposite directions, consistent with redistribution of stored ions fromone energy minimum to the other across the plasma membrane. Furthermore,the induced capacitance dq/dV from the oxonol movement is calculated tobe ˜5 pF for 100 mV depolarization. This value corresponds to roughlyone third the membrane capacitance without the dye. Interestingly,sodium channel gating charges are also responsible for about 33% of thetotal capacitance of squid axons for small depolarizations [Hodgkin, A.1975. The optimum density of sodium channels in an unmyelinated nerve.Philos. Trans. R. Soc. Lond. [Biol] 270:297–300]. Negligible currentswere observed in the absence of the oxonol. DiSBA-C₁₀-(3) gavedisplacement currents of approximately the same speed, whereas analogueswith R=butyl and ethyl gave much slower currents. The butyl compound hada time constant of ˜18 ms and the currents from the ethyl compound werevery small, slow, and difficult to observe.

FIGS. 4 and 5 show the voltage dependence and time constants for chargetranslocation in a cell loaded with about 4 times as much oxonol as inthe experiment of FIG. 3. In FIG. 4, the circles are the data from theon response and the squares from the tail currents. The raw data werefit to a single exponential and the charge moved, the area, wascalculated as the product of the current amplitude and the timeconstant. The experimental data are in reasonable accord with existingmodels of hydrophobic ion transport between two energy minima near theaqueous interfaces of the lipid bilayer [Ketterer, B, Neumcke, B., andLäuger, P. 1971 Transport mechanism of hydrophobic ions through lipidbilayer membranes J. Membrane Biol. 5:225 245; Andersen, O. S. andFuchs, M. 1975. Potential energy barriers to ion transport within lipidbilayer. Biophys. J. 15:795–830; Benz, R., Läuger, P., and Janko, K.1976. Transport kinetics of hydrophobic ions in lipid bilayer membranes.Biochim. Biophys. Acta 455:701–720]. These models predict that theequilibrium charge displacement q(V) and the translocation time constant(V) should depend on the externally applied membrane potential V in thefollowing manner:

$\begin{matrix}{{\Delta\;{q(V)}} = {\Delta\; q_{\max}{\tanh\left\lbrack \frac{q\;{\beta\left( {V - V_{h}} \right)}}{2{kT}}\; \right\rbrack}}} & (1) \\{{\tau(V)} = {\tau_{\max}{{sech}\left\lbrack \frac{q\;{\beta\left( {V - V_{h}} \right)}}{2{kT}}\; \right\rbrack}}} & (2)\end{matrix}$

V_(h), the membrane potential at which there are equal numbers of ionsin each potential energy well, could differ from zero because ofmembrane asymmetry. β is the fraction of the externally appliedpotential effectively felt by the translocating ion; q is the charge oneach ion, k and T are Boltzmann's constant and absolute temperature.q_(max) and _(max) are respectively the total charge in each energy welland the time constant for translocation, both at V=V_(h). The smoothcurve in FIG. 4 is the fit to Eq. 1 with q_(max)=4770±140 fC,β=0.42±0.02, and V_(h)=−3.8±1.5 mV. Likewise the smooth curve in FIG. 5is the fit to Eq. 2 with _(max)=2.9 ms at V_(h)=−5 mV and β=0.42.

These results demonstrate that the oxonol senses a significant part ofthe electric field across the membrane, that it translocates in ˜3 ms orless, and that the greatest sensitivity and linearity of translocationas a function of membrane potential is in the physiologically relevantrange.

To transduce charge displacements into optical signals, the oxonolfluorescences at the intracellular and extracellular membrane bindingsites is made different. Fluorescence asymmetry is created with theintroduction of fluorescently labeled lectins bound to the extracellularmembrane surface. Excitation of FL-WGA leads to energy transfer tooxonols located in the extracellular membrane binding site as shown inFIG. 1. The extinction coefficient and the fluorescence quantum yield ofFL-WGA were measured to be 222,000 M⁻¹cm⁻¹ (˜3 fluorescein/protein) and0.23, respectively. In Jurkat cell suspensions labeled with FL-WGA, upto 30% of the lectin fluorescence intensity was quenched upon titrationof DiSBA-C₄-(3). In the best case where all of the quenching is due toenergy transfer, the average distance from the lectin to themembrane-bound oxonol is still greater than 50 Å, the calculated Försterdistance R_(o) for the FL-WGA/oxonol pair. The spectral overlap betweenthe FL-WGA emission and DiSBA-C₆-(3) excitation is given in FIG. 2.Because FRET falls off with the inverse sixth power of the distanceseparating the two fluorophores, energy transfer to oxonols in theintracellular membrane site, an additional 40 Å away, is probablynegligible.

Upon depolarization, the oxonol molecules redistribute such that moreare bound to the intracellular site and less to the extracellular one.This change manifests itself with a decrease in the energy transfer,resulting in an increase in the fluorescence of the FL-WGA and aconcomitant decrease in the oxonol emission. The fluorescence signals ina voltage clamped L-M(TK⁻) cell labeled with (the DiSBA-C₄-(3)/FL-WGApair) and depolarized with four increasing voltage steps are shown inFIG. 6. The data are the average of 29 sweeps. The FL-WGA emissionincreases 7–8%, the oxonol fluorescence decreases 10% and theFL-WGA/oxonol emission ratio changes 19% for a 120 mV depolarization.The simultaneous changes in donor and acceptor emissions is consistentwith the FRET mechanism outlined in FIG. 1. The decrease in oxonolemission with depolarization is opposite to what is observed for theslow voltage-sensitive uptake of oxonols in cells [Rink et al. 1980,supra]. The fluorescence changes have time constants of ˜18 ms at 20 C,in agreement with the DiSBA-C₄-(3) displacement currents. No largefluorescence changes are observed in the absence of FL-WGA. Thetranslocation rate of DiSBA-C₄-(3) speeds up with increasingtemperature. The time constant falls to 7–8 ms at 29 C, corresponding toan activation energy of ˜17 kcal/mol. However, raising the temperaturealso increases internalization of the lectin and eventually decreasesthe fluorescence change. The oxonols with R=ethyl and butyl also reachinternal cellular membranes, though active membrane internalization isprobably not necessary. Additional dilution of the voltage-dependentFRET signals arises from spectral overlap of the fluorescein and oxonol,such that some of the light in the fluorescein emission channel comesfrom the oxonol and vice versa.

Increasing the length of the alkyl chains on the oxonol improves theresponse times significantly. The DiSBA-C₆-(3)/FL-WGA pair, has a timeconstant of ˜3 ms at 20 C, while the DiSBA-C₁₀-(3)/FL-WGA pair, respondswith a time constant of 2 ms, as shown in FIG. 7. The solid curve is afit to a single exponential with a 2 ms time constant. The data is theaverage of 3 L-M(TK⁻) cells, at 20 C, acquired at 2 kHz. The response inthe figure is slightly slower than the true value because of smoothing.The fluorescence time constants are in agreement with those from thedisplacement currents, for example in FIG. 3. The beneficial effect ofadding hydrophobicity to the oxonol in the form of longer alkyl chainsreaches a plateau. There is a large 6-fold increase in translocationrate substituting hexyl for butyl on the oxonol core. However, additionof twice as many methylene groups in going from the hexyl to the decylcompound results in less than a 2-fold increase. These fastertranslocating oxonols are essentially insoluble in water and requiremodified procedures to load into cells. DiSBA-C₆-(3) is easily loaded innormal medium supplemented with 1.5 mM β-cyclodextrin to complex thealkyl chains. Nonfluorescent DiSBA-C₆-(3) aggregates in Hanks BalancedSalt Solution (HBSS) become fluorescent upon addition of β-cyclodextrin.DiSBA-C₁₀ (3) requires loading in a medium of low ionic strength withosmolarity maintained with sucrose. Labeling is confined almostexclusively to the plasma membrane, presumably because thehydrophobicity is now great enough to prevent desorption from the firstmembrane the dye encounters.

Example V—Measurement of Membrane Potential with Oxonol Dyes as FRETAcceptors and Fluorescent Lipid FRET Donors

A. Trimethine Oxonols

A 6-chloro-7-hydroxycoumarin conjugated todimyristoylphosphatidylethanolamine (DMPE) via a glycine linker, Cou-PE,has been prepared and found to function as an excellentvoltage-sensitive FRET donor tobis-(1,3-dialkyl-2-thiobarbiturate)-trimethineoxonols. This new FRETpair has given an 80% ratio change for a 100 mV depolarization in anastrocytoma cell, which is the largest voltage-sensitive optical signalobserved in a cell, FIG. 17. The voltage sensitivity of this FRET pairis consistently 2–3 times better than Fl-WGA/trimethineoxonol in avariety of cell types. In L-cells, ratio values between 25–50% arefound, with equal percent changes found in both channels, FIG. 18. Inneonatal cardiomyocytes, 5–30% fluorescence ratio changes were observedfor spontaneously generated action potentials. The largest signals arealmost 4 times larger than those possible with Fl-WGA/trimethineoxonol.An example of such a large change from a single cluster of heart cellsis given in FIG. 19. The benefits of a ratio output are evident in thefigure. The individual wavelengths, at the top of the figure, show adecreasing baseline that is due to fluorophore bleaching. The ratio datashown on the bottom of the figure compensates for the loss of intensityin both channels and results in a flat baseline. Furthermore, motionartificially causes broadening of the individual wavelength responses.The ratio data reduces these artifacts and results in a sharper signalthat more closely represents the actual voltage changes. The greatersensitivity for this new FRET pair is most likely due to a combinationof factors. Moving the donor closer to the membrane surface anddecreasing the Forster transfer distance, R_(o), may result in increasedFRET discrimination between the mobile ions on the same and oppositesides of the membrane. Also, the increased spectral separationfacilitates collection of the donor and acceptor emission and reducessignal loss due to crosstalk.

B. Pentamethine Oxonols

Bis-(1,3-dialkyl-2-thiobarbiturate)-pentamethineoxonols have beenprepared by condensing 1,3 substituted thiobarbituric acids withglutacondialdehyde dianil monohydrochloride, also known asN-[5-(phenylamino)-2,4-pentadienylidene]benzenamine monohydrochloride.The pentamethine oxonols absorb at 638 nm (ε=225,000 M⁻¹ cm⁻¹) andfluoresce maximally at 663 nm in ethanol. The absorbance and emissionare shifted 100 nm to longer wavelengths, compared to the trimethineoxonols. This shift is consistent with other polymethine dyes, such ascyanines, where addition of 2 additional methine units results in 100 nmwavelengths shifts to the red.

The pentamethines can be loaded into cells in culture in the same manneras the trimethine oxonol. The butyl compound, DiSBAC₄(5), can be loadedin Hanks' Balanced Salt Solution, while the hexyl compound, DiSBAC₆(5),requires addition of beta-cyclodextrin to mask the hexyl side chains andsolubilize the hydrophobic oxonol.

Voltage-sensitive FRET from various plasma membrane-bound fluorescentdonors to the pentamethine oxonols have been demonstrated in singlecells. Fl-WGA has been shown to undergo FRET with the pentamethine andgive ratio changes comparable to those observed for the trimethineoxonol. In astrocytoma cells, ratio changes of 15–30% were recorded fora 100 mV depolarization step from −70 mV. Apparently, the decrease inFRET due to the reduced overlap integral, J, on moving the oxonolabsorbance 100 nm longer is compensated by increased selectivity of FRETto the extracellular face of the membrane relative to the intracellularone and/or decreased spectral overlap.

Fluorescent phosphatidylethanolamine conjugates have also been found tofunction as FRET donors to the pentamethine oxonol. The structures of PEconjugates tested are shown in FIG. 20. NBD-PE/pentamethineoxonol pairhas given 1–10% ratio changes per 100 mV. Cou-PE/pentamethineoxonol hasgiven 15–30% ratio changes in voltage clamped astrocytomas for 100 mVdepolarization. This pair is remarkable because the Cou-PE emission andDiSBAC₆(5) absorbance maxima are separated by 213 nm and there is hardlyany visible overlap, FIG. 21. The large extinction at long wavelengthsof the pentamethine enable FRET between the coumarin and thepentamethine oxonol. The R_(o) for this pair has been calculated to be37 Å, using a quantum yield value of 1.0 for the Cou PE.

The membrane translocation rates for the pentamethines are 5–8 timesfaster than the trimethine analogues. DiSBAC₄(5) displacement currentsin voltage-clamped astrocytoma cells showed that butyl pentamethineoxonol jumps across the plasma membrane with a time constant of ˜2 ms inresponse to voltage steps of +20–120 mV. The trimethine analoguetranslocates ˜18 ms under identical conditions. The displacementcurrents of DiSBAC₆(5) decay very rapidly and are difficult to separatefrom the cell capacitance. As a result of the large voltage-dependentsignal from the Cou-PE/DiSBAC₆(5) pair, it was possible to opticallymeasure the speed of voltage response of DiSBAC₆(5). The time constantfor DiSBAC₆(5) translocation was measured optically at 0.383±0.032 ms inresponse to a 100 mV depolarization step, using FRET from asymmetricallylabeled Cou-PE. The ratio data and the exponential response are shown inFIG. 22. The enhanced translocation rates result from the greater chargedelocalization and slightly more hydrophobicity of the pentamethineoxonols. The rapid voltage response of DiSBAC₆(5) is the fastest knownfor a permeant, highly fluorescent molecule. The submillisecond responseis fast enough to accurately register action potentials of singleneurons.

Example VI—Measurement of Membrane Potential with Oxonol Dyes as FRETDonors

The direction of energy transfer can be reversed using TR-WGA instead ofFL-WGA. DiSBA-C₆-(3) functions as a FRET donor to TR-WGA in L-M(TK⁻)cells with the same response time as FL-WGA/DiSBA-C₆-(3). The spectraloverlap of this FRET pair is shown in FIG. 2. The signal change,however, is only one half that for FL-WGA/DiSBA-C₆-(3).

DiSBAC₆(3) has been successfully used as a FRET donor to Cy5 labeled PE,in B104 neuroblastoma cells. The ratio changes of 5–15%/100 mV are thelargest observed with the mobile ion being the donor.

Example VII—Measurement of Membrane Potentials in Different Cell Types

The FL-WGA/DiSBA-C₆-(3) system was tested in a variety of cell lines. Inneonatal cardiac myocytes, the two fluorophores could be loaded withoutaffecting the spontaneous beating. Therefore, the added capacitance fromthe oxonol displacement current did not prevent generation of actionpotentials. The periodic 90 mV action potentials [Conforti, L, Tohse,N., and Sperelakis, N. 1991. Influence of sympathetic innervation on themembrane electrical properties of neonatal rat cardiomyocytes inculture. J. Devel. Physiol. 15:237–246] could be observed in a singlesweep, FIG. 8C. The ratio change without subtraction of any backgroundfluorescence was 4–8%. Motion artifacts were observed in the singlewavelength data. In FIGS. 8A and B, large slow changes in the detectedlight were observed in both channels. Satisfyingly, these effects wereessentially eliminated in the ratio data. The data were acquired at 100Hz and 10 M of isoproterenol was added to the bath solution. The voltagedependent fluorescence changes are faster than the mechanically basedartifacts, as expected [Hill, B. C. and Courtney, K. R. 1982.Voltage-sensitive dyes discerning contraction and electrical signals inmyocardium. Biophys. J. 40:255–257]. Some cells, loaded with oxonol at2.3 M, did stop beating after about 7 seconds of continuous exposure tothe xenon arc illumination. At 0.6 M loading, the phototoxicity andunfortunately the signal were reduced. In differentiated B104neuroblastoma cells an 8% ratio increase was recorded, without anybackground subtraction, for a 120 mV depolarization. The inward sodiumcurrents did not deteriorate from phototoxic effects during experimentswith excitation exposures totaling 10–20 s. FL-WGA/DiSBA-C₆-(3) labeled1321N astrocytoma cells showed oxonol and FL-WGA fluorescence almostexclusively on the plasma membrane. Ratio changes of 22–34% for 100 mVwere observed in photometry experiments such as FIG. 9. After a 50 msdelay, the membrane potential was depolarized 100 mV from −70 mV for 100ms. The traces are the average of 4 sweeps acquired at 300 Hz, with nosmoothing. The time constant for the fluorescence changes is less than3.3 ms consistent with the displacement currents, such as those in FIG.3. A small background signal was subtracted from the starting signal,<5% for the oxonol channel and <15% for the fluorescein channel. Thefluorescence intensities in the fluorescein and oxonol channelsincreased ˜17% and decreased ˜16% respectively for 100 mVdepolarization. In these cells, unlike the L-M(TK⁻), the crosstalkbetween emission channels was decreased and larger changes occurred inthe fluorescein signal. These signal changes are the largest millisecondmembrane potential dependent ratio changes observed in single cells.Previous investigations have shown that 4-ANEPPS gives a 9%/100 mVexcitation ratio change [Montana, V., Farkas, D. L., and Loew, L. M.1989. Dual-wavelength ratiometric fluorescence measurements of membranepotential. Biochemistry 28:4536–4539]. In addition, FL-WGA/DiSBA-C₆-(3)fluorescence changes in each emission channel are comparable to thelargest reported changes, for example, the 21% /100 mV change in aneuroblastoma using RH-421 [Grinvald, A., Fine, A., Farber, I. C., andHildesheim, R. 1983. Fluorescence monitoring of electrical responsesfrom small neurons and their processes. Biophys. J. 42:195–198]. Thelarge signals from FL-WGA/DiSBA-C₆-(3) made it possible to record ratioimages of membrane potential changes in voltage clamped L-M(TK⁻) andastrocytoma cells using a high speed confocal microscope.

The astrocytoma cells gave a 10–20% ratio increase that was localized tothe plasma membrane for a 120 mV depolarization.

Example VIII—Synthesis of Fluorescent Tetraaryl Borates

With reference to FIG. 10, in a 25 mL flame dried two neck flask 0.788 g(580 L, 2.2 mmol) of compound 1 was dissolved in 6 mL of dry hexaneunder argon. After cooling the flask to −70 C, 1.46 mL of a 1.5 Mn-butyllithium solution (2.2 mmol) was added via syringe. In a separateflask 0.60 dry borane II was dissolved in a deoxygenated mixture of 6 mLhexane and 1.5 mL of freshly distilled THF. The borane solution was thenadded via syringe to the lithium reagent. A solid immediatelyprecipitated. After 30 min the cold bath was removed and the reactionwas allowed to slowly heat up. Three hours later the solvent wasdecanted off and the solid rinsed with more hexane. The solid wasdissolved in acetonitrile and water and then poured into a separatoryfunnel. The aqueous layer was again washed with hexane and thenextracted with ethyl acetate. Half of the extract was concentratedyielding 124.9 mg (170.5 mol) of the desired product. This product wasthen mixed with 97 mg of tetrabutylammonium fluoride in acetonitrile for15 min at room temperature. After workup 129.1 mg of compound III as thetetrabutylammonium salt was recovered (93%). ¹HNMR (d₆ acetone) 7.61 (brd, 2H, CF₃-phenyl group), 7.43 (cm, ˜3H, CF₃-phenyl group), 6.90–7.26(cm, ˜7H, CF₃-phenyl group), 3.43 (cm, 8H, NCH ₂CH₂CH₂CH₃), 1.81 (cm,˜8H, NCH₂ CH₂CH₂CH₃), 1.42 (cm, ˜8H, NCH₂CH₂ CH ₂ CH₃), 0.93 (t, J=7.1Hz, NCH₂CH₂CH₂CH ₃).

With reference to FIG. 10, for synthesis of compound IV, in a 5 mL roundbottom flask 14 mg (17.2 umol) of compound III, 7 mg (25.8 umol) ofbromomethylbimane, 27.3 mg (172 umol) of potassium carbonate, and 5 mg(18.9 umol) of 18-crown-6 were mixed in 0.6 mL of dry acetonitrile. Themixture was heated at 70 C 1.5 h. After cooling, the reaction mixturewas dissolved in ethyl acetate and washed 3× with water. The organicresidue was purified by flash chromatography eluting withtoluene/acetone (2:1). The major band was collected yielding 12.1 mg(70%) of pure product IV tetrabutyl ammonium salt. ¹HNMR (d₆ acetone)7.58 (br d, 2H, CF₃-phenyl group),7.4–7.5 (cm, 2H, CF₃-phenyl group),7.0–7.3 (cm, ˜10H, CF₃-phenyl group), 5.29 (d, J=1.6 Hz, 2H, CH₂), 3.46(cm, 8H, NCH ₂CH₂CH₂CH₃), 2.56 (d, J=0.7 Hz, 3H, bimane methyl), 1.84(cm, ˜8H, NCH₂ CH₂CH₂CH₃), 1.79 (d, J=0.8 Hz, 3H, bimane methyl), 1.76(s, 3H, bimane methyl), 1.44 (cm, ˜8H, NCH₂CH₂CH ₂CH₃), 0.98 (t, J=7.2Hz, NCH₂CH₂CH₂CH ₃); _(f)=0.73 in dioxane based on quinine sulfate in0.1 N H₂SO_(4 f)=0.55.

Example IX—Synthesis of Asymmetric Oxonols with a Linker Group

(A)This example illustrates the synthesis of asymmetric oxonolscontaining a built-in linker group. With reference to FIG. 11, forsynthesis of compound V, 4.35 g (21.7 mmol) of 1,12-diaminododecane wasdissolved in 40 mL of dry CH₂Cl₂. Via syringe, 2.62 mL (2.17 mmol) ofbutyl isothiocyanate was added to the reaction flask. A white solid hadprecipitated after 15 minutes. One hour after the addition, the reactionmixture was filtered. The filtrate was then evaporated leaving a whitesolid. The solid was redissolved in 45 mL of dry CH₂Cl₂ and mixed with2.44 mL of N,N-diisopropylethylamine (DIEA) and 3.9 g (17.9 mmol) ofdi-tert-butyl dicarbonate. After reacting for 1 hour, the mixture waspoured into a separatory funnel and washed with 5% sodium bicarbonate. Asolid came out of solution and was filtered away (<100 mg). The organicsolution was then washed with water and a saturated brine solution. Theorganic layer was then dried with MgSO₄ and filtered. The filtrate wasevaporated leaving a white solid, which was recrystallized in isopropylether yielding 4.30 g (10.3 mmol) of pure compound V (48% overall).¹HNMR (CDCl₃) 5.73 (br s, 2H, thioamide), 4.50 (br s, 1H, carbamate),3.40 (br s, 4H, NCH ₂), 3.10 (q, J=7.2 Hz, ˜3H, CH ₂ next to carbamate),1.44 (s, 9H, t-butyl), 1.2–1.7 (cm, bulk CH ₂ s), 0.94 (t, J=7.2 Hz,n-butyl methyl).

For preparing compound VI, 441 mg (19.2 mmol) of sodium was dissolved in5 mL of dry ethanol under argon. When almost all of the sodium wasdissolved, 2.92 mL (3.1 g, 19.2 mmol) of diethyl malonate was added tothe ethoxide solution. Some solid precipitated out of solution. 4.0 g(9.6 mmol) of compound V was added and the mixture was refluxed underargon at 100 C for 70 hours. After cooling, the reaction mixture wasfiltered and washed with ethanol. Water was added to the filtrate and awhite solid precipitated out of solution. The solid (779 mg, mostly ofunreacted starting material) was filtered away. The filtrate was thenacidified to pH ˜2 and then extracted into ethyl acetate. The organiclayer was then dried with MgSO₄ and filtered. After removing thesolvent, 1.6 g (3.3 mmol) of a yellow oil was recovered (34%) ¹HNMR(CDCl₃) 4.22 (cm, 4H, NCH ₂ next to barbiturate). 3.63 (s, 2H, ring CH₂), 2.99 (cm, 2H, CH ₂ next to carbamate), 1.53 (cm, 4H, NCH₂CH ₂), 1.34(s, 9H, t-butyl), 1.1–1.3 (cm, bulk CH ₂ s), 0.85 (t, J=7.4 Hz, n-butylmethyl).

To prepare compound VII, 1 mL of trifluoroacetic acid (TFA) was addedwith stirring to 200 mg (0.41 mmol) of compound VII dissolved in 3 mL ofCH₂Cl₂. After 1.25 hours, all the solvent was removed under reducedpressure. One equivalent each ofN-[5-(phenylamino)-2,4-pentadienylidene]aniline monohydrochloride and1,3-di-n-butylthiobarbiturate was added and all three componentsdissolved in 1 mL pyridine and left overnight. The product was purifiedfrom the other pentamethine oxonols by flash chromatography. Thenonpolar products were eluted with CHCl₃/CH₃OH (9:1). The pure productcontaining the linker eluted with CHCl₃/CH₃OH (1:1). The product wasbound very tightly to the silica gel and only 10 mg of product wasrecovered. ¹HNMR (CDCl₃/CD₃OD) 7.5–7.8 (cm, 4H, vinyl methines), 7.35(t, J=˜14 Hz, 1H, central methine), 4.34 (br t, ˜10 H, NCH ₂ next tobarbiturate), 2.72 (cm, ˜3H, CH ₂ next to amine), 1.4–1.7 (br cm, ˜12H,NCH₂CH ₂), 1.0–1.4 (cm, ˜40H, bulk CH ₂ s), 0.81 (t, J=7.3 Hz, 9H,n-butyl methyl).

(B)This particular example is with reference to FIGS. 15 and 16.

N-butyl-N-5-pentanol thiourea (6):5-amino-1-pentanol (1.416 mL, 13 mmol)was dissolved in 7 mL of CH₂Cl₂. Under argon, butylisothiocynate (1.570mL, 13 mmol) was added via syringe. After 2.5 h, the solvent was removedunder vacuum leaving an oil. Under high vacuum, the oil was freeze dried2× with liquid nitrogen and left under reduced pressure over night. Thenext morning 2.615 g of pure solid product was collected (12 mmol, 92%).¹H NMR (CDCl₃): d 5.90 (br s, 2H, NH), 3.66 (t, J=6.2 Hz, 2H, RCH ₂OH),3.42 (br m, 4H, NHCH₂R), 1.80 (br s, 1H, OH), 1.3–1.7 (unres. cm's, 10H,bulk methylenes), 0.94 (t, J=7.2 Hz, 3H, methyl). ¹³C NMR (CDCl₃): d181.5 (thiocarbonyl), 62.2 (RCH₂OH), 44.2 (NHCH₂R), 44.1 (NHCH₂R), 31.9(CH₂), 31.0 (CH₂), 28.6 (CH₂), 23.0 (CH₂), 19.9 (CH₂), 13.6 (CH₃).

1-butyl,3-(5-pentanol) thiobarbiturate (7):In dry EtOH, 345 mg (15 mmol)of Na was dissolved. After almost all of the Na had dissolved,diethylmalonate (2.278 mL, 15 mmol) was added under argon. The mixturewas then heated to 60° C. to dissolve the precipitated sodium malonate.The heat was then removed and N-butyl N-5-pentanol thiourea (6) (1.310g, 6 mmol) was added. The reaction mixture was refluxed a 100° C. for3.5 days. After cooling, the reaction mixture was filtered and washedwith EtOH. An approximately equal volume of H₂O was added to thefiltrate and acidified to pH 1–2 with conc. HCL. The aqueous solutionwas extracted 3× with 1:1 EtOAc/hexanes. The combined extracts weredried with MgSO₄, filtered, and concentrated leaving an oil. TLCEtOAc/MeOH (4:1) showed that in addition to the major barbiturateproduct there were two nonpolar impurities. Flash silica gelchromatography afforded some purification (4×17 cm), eluting withEtOAc/MeOH (4:1). The material was still an oil and a second column wasdone eluting with CHCl₃/MeOH/AA (90:8:2). Despite being an oil, 0.726 g(2.54 mmol, 42%) of pure product was recovered. ¹H NMR (CDCl₃): d 4.31(cm, 4H, NCH₂R), 3.71 (br s, 2H, ring methylene), 3.63 (t, J=6.3 Hz, 2H,RCH₂OH), 2.75 (br s, 1H, OH), 1.5–1.8 (cm, 6H, bulk methylenes), 1.2–1.5(cm, 4H, bulk methylenes), 0.94 (t, J=7.2 Hz, 3H, methyl).

1-butyl,3-(5-bromopentane) thiobarbiturate (8): (7) (98 mg, 343 umol)was dissolve in 600 uL dry CH₂Cl₂ and mixed with carbon tetrabromide(142 mg, 429 umol). The solution was cooled on ice andtriphenylphosphine (135 mg, 515 umol) was added. The solution bubbledand turned yellow immediately. After 30 min. the solvent was removed andhexane was added to the solid residue. The mixture was allowed to stirovernight. TLC showed only 1 barbiturate in hexane solution along withtriphenylphospine oxide. The impurity was removed by flash silica gelchromatography (2.5×22 cm) packed in EtOAc/MeOH (98:2). The nonpolarimpurity was eluted off the column using the packing solvent followed byEtOAc/MeOH (90:10). The desired product was eluted with CHCl₃/MeOH/AA(93:5:2), yielding 40 mg (115 umol, 34%).

¹H NMR (CDCl₃): d 4.33 (cm, 4H, NCH₂R), 3.72 (s, 2H, ring methylene),3.42 (t, J=6.7 Hz, 2H, RCH₂Br), 1.91 (cm, 2H, methylene), 1.66 (cm, 4H,methylenes), 1.52 (cm, 2H, methylene) 0.95 (t, J=7.2 Hz, 3H, methyl).

1,3-di-butyl-5-(3-phenylamino propendienyl) thiobarbiturate(10):Malonaldehyde bis(phenylimine) (500 mg, 1.69 mmol) was dissolved in20 mL of dry DMF. Separately, 1,3 di-butyl thiobarbiturate (430 mg, 1.76mmol) was dissolved in 5 mL dry pyridine and placed in a 10 mL droppingfunnel. The thiobarbiturate solution was slowly added over 5 min and thereaction was left stirring for 5 h. About 20 mL of water was added tothe mixture and a yellow solid precipitated out of solution. The solidwas filtered and dried yielding 575 mg (1.5 mmol, 89%). Minorimpurities, including oxonol, were removed by flash silica gelchromatography (3×15 cm) eluting with EtOAc/hexanes (1:1). Some materialprecipitated on the column. Nevertheless after drying, 390 mg of pureproduct was recovered (1 mmol, 59%). ¹H NMR (CDCl₃/MeOH): d 8.10 (d,J=3.0 Hz, 1H), 8.04 (s, 1H), 7.3–7.5 (cm, 3H), 7.1–7.25 (cm, 3H), 4.40(cm, 4H, NCH₂R), 1.65 (cm, 4H, NCH₂CH₂R), 1.36 (cm, 4H, NCH₂CH₂CH₂CH₃),0.90 (t, J=7.3 Hz, 6H, methyls).

1-(1,3-dibutylthiobarbiturate)-3-(1-butyl,3-(5-hydroxypentyl)thiobarbiturate)trimethineoxonol triethylammonium salt (11):Compound (7) (85 mg, 297umol) was mixed with (10) (114 mg, 297 umol) in 1.2 mL dry pyridine andleft stirring for 17 h. TLC EtOAc/MeOH showed that there was 3 majoroxonol products. Conc. HCL was added to 80% of the reaction mixturewhich caused a solid to precipitate out of solution. The solid wasfiltered and washed with water. After drying 220 mg of red solid wasrecovered. 96 mg of this solid was mixed with 1–2 mL EtOAc and filtered.The remaining solid was dissolved in CHCl₃/MeOH (95:5) and loaded on toa 19×2.5 cm silica gel column. Eluting with the same solvent, the mostnonpolar oxonol was eluted off the column. The solvent was then changedto CHCl₃/MeOH/Et₃N (90:8:2) to elute the middle band which was shown byNMR to be the desired product. After concentrating and drying, 18.5 mg(28 umol, 27%) of the pure product was recovered. ¹H NMR (CDCl₃): d 8.60(t, J=13.9 Hz, 1H, central methine), 8.14 (dd, J₁=13.8 Hz, J₂=2,2 Hz,2H, methines), 4.45 (cm, 8H, NCH₂R), 3.66 (t, J=6.3 Hz, 2H, RCH₂OH),3.20 (q, J=7.3 Hz, 6H, triethylammonium), 1.5–1.8 (cm, 8H, NCH₂CH₂R),1.2–1.5 (cm, 10H, bulk methylenes), 1.35 (t, J=7.3 Hz, 9H,triethylammonium), 0.95 (t, J=7.2 Hz, 9H, terminal methyl). MS.

1-(1,3-dibutylthiobarbiturate)-3-(1-butyl-3-(5-tosyloxypentyl)thiobarbiturate)trimethineoxonol (12):In 4 mL pyridine, (11) (86.1 mg, 131 umol) wasmixed with tosyl chloride (333 mg, 1.75 mmol). The reaction mixture wasstirred for 3.5 h before the solvent was removed under vacuum. Theresidue was dissolved in EtOAc and washed with 1 M HCL, followed by sat.brine 2×. TLC EtOAc/MeOH (9:1) showed that most of the starting materialwas converted to a more non polar product. However, a polar oxonolimpurity was evident and the material had to be further purified byflash chromatography. The column was packed in CHCl₃/MeOH (97:3). It wasnecessary to increase the polarity to CHCl₃/MeOH (92:8) in order toelute off the product. The fractions containing the desired product werecombined and dried, yielding 74.8 mg (102 umol, 78%) of product. ¹H NMR(CDCl₃): d 8.50 (t, 1H, central methine), 7.92 (dd, J₁=14 Hz, J₂=2 Hz,2H, methines), 7.81 (d, 2H, tosyl), 7.35 (d, J=8.2 Hz, 2H, tosyl), 4.37(cm, 8H, NCH₂R), 4.09 (br t, 2H, RCH₂OTs), 2.45 (s, 3H, tosyl methyl),1.2–1.9 (unres. cm, bulk methylenes), 0.91 (t, J=7.2 Hz, 9H, terminalmethyl).

Example IX—Synthesis of Fluorescent Lanthanide Chelates with a SingleNegative Charge

Terbium(III) Bis-(N,N′-bis(salicylidene)ethylenediamine) piperidiniumsalt, Hpip⁺ Tb(SALEN)₂-:N,N′-bis(salicylidene)ethylenediamine (SALEN)(0.719 g, 2.68 mmol) was dissolved in 40 mL MeOH at 60° C. Terbiumchloride hexahydrate (0.5 g, 1.34 mmol) dissolved in 1 mL water wasadded to the solution. Piperidine (536 μL, 5.42 mmol) was added and ayellow precipitate immediately formed. After 1 h, the heat was removedand the reaction mixture was left stirring overnight. The solid wasfiltered and dried yielding 709 mg (0.91 mmol, 68%) of the desiredcomplex. Electrospray (neg. ion) MS [MeOH/H₂O: 95/5] (peak, rel. int.)691.2 (M⁻¹, 100) calc. M⁻¹=691.5 amu.

Europium(III) Bis-(N,N′-bis(salicylidene)ethylenediamine) piperidiniumHpip⁺ Eu(SALEN)₂-:N,N′-bis(salicylidene)ethylenediamine (SALEN) (0.360g, 1.34 mmol) was dissolved in 40 mL MeOH and piperidine (298 μl, 3.02mmol) at 60° C. Europium chloride hexahydrate (0.246 g, 0.67 mmol)dissolved in 0.5 mL water was added to the solution and a yellowprecipitate immediately came out of solution. After 1 h, the heat wasremoved and the reaction mixture was left for 2 hours. The solid wasfiltered and dried yielding 341 mg (0.44 mmol, 66%) of the desiredcomplex.

Example X—Construction of Targeted Naturally Fluorescent Proteins

Molecular Biology. The Aequorea victoria GFP coding sequence (Clontech)was engineered to contain the mutations S72A, Y145F and T203I (Sapphire)(SEQ. ID. NO: 1) (Green Fluorescent Proteins, Chapter 2, pages 19 to 47,edited Sullivan and Kay, Academic Press). This fluorescent protein hasan excitation peak around 395 nm and an emission peak around 512 nm,making it a useful donor with the trimethine oxonol acceptor. To createa targetable construct, the plasma membrane localisation signal form thetyrosine kinase lyn was fused in frame with the Sapphire codingsequence. This was achieved using two stage PCR by adding the sequence(MGCIKSKRKDNLNDDGVDMKT, SEQ. ID. NO: 15) to the N-terminus of Sapphire.This sequence, which is derived from the first 21 amino acids of lyn,contains a well defined membrane localization domain that serves in vivoto specifically localize lyn to the inner plasma membrane of mammaliancells.

In the first-stage PCR, a sense primer(ATTCCCAAGCTTGCGGCCGCCACCATGGGCTGCATCAAGAGCAAGCGCAAGGACAACCTGAACGACGACGGCGTG, SEQ ID. NO: 20) and an anti-sense primer(CCGGAATTCTTACTTGTACAGCTCGTCCATGCC, SEQ ID. NO: 21) were used. In thesecond-stage, a sense primer(GACAACCTGAACGACGACGGCGTGGACATGAAGACCATGGTGAGCAAGGGC GAGGAGCTG SEQ ID.NO: 22) and the anti-sense primer were used. The PCR product wasdigested with HindIII and EcoRI, and ligated into the mammalianexpression vector pcDNA3 (Invitrogen) Additional targeting sequenceswere made based on this construct. To utilize the NcoI sites that flankthe Lyn sequence, the Lyn Sapphire insert was subcloned from pcDNA3 intopBluescriptII (Stratagene) using the restriction sites HindIII andEcoRI. Oligonucleotides coding for the additional targeting sequencesLyn D10, D15 and RRR (see Table 4) with their complimentary strands weresynthesized, using standard techniques.

TABLE 4 Construct Name Sequence SEQ. ID. NO: Orientation Lyn SapphireMGCIKSKRKDNLNDDGVDMKT SEQ. ID. NO: 15 N-terminal Lyn D15 SapphireMGCIKSKRKDNLNDDT SEQ. ID. NO: 17 N-terminal Lyn D10 Sapphire MGCIKSKRKSEQ. ID. NO: 18 N-terminal Lyn RRR Sapphire MGCIKSKRKDNLNDDRRRT SEQ. ID.NO: 19 N-terminal Ras Sapphire KKKKKKKSKTKCVIM SEQ. ID. NO: 16C-terminal LynRas Sapphire MGCIKSKRKDNLNDDGVDMKT + SEQ. ID. NO: 15N-terminal + KKKKKKKSKTKCVIM SEQ. ID. NO: 16 C-terminal

These oligomers were phosphorylated using T4 polynucleotide kinase,hybridized by heating the complimentary strands to 95° C. and thenslowly cooling the is samples to room temperature. Double strandedoligomers, containing the required targeting sequence, were inserted 5′of the sapphire GFP coding region after removal of the original Lynsequence by digestion of the lyn Sapphire DNA with NcoI. The orientationand sequence of the inserts was confirmed by DNA sequencing.

To generate fluorescent proteins with C-terminal localization motifs andC and N-terminal localization motifs, oligonucleotides was synthesizedand used as an antisense primer in PCR. The ras oligonucleotidecontained the antisense sequence of the C-terminal CAAX-box andpolybasic region of K-Ras followed by 16 nucleotide bases that overlapinto the C-terminus of Sapphire GFP. The T3 primer was used as the senseprimer. Either Lyn Sapphire or Sapphire alone plasmids were used astemplate DNA, depending on the constructs required. All inserts weresubcloned into the mammalian expression construct pcDNA3 prior totransfection into cells.

The first requirement for using GFP as a voltage-sensitive FRET donor isto selectively target it to the desired membrane, in this example theplasma membrane. While many proteins bind to the cell membrane, a largenumber of these candidate proteins also have high expression levels inother cellular locations. Often fusion of a naturally fluorescentprotein to a protein that is expressed at the plasma membrane willresult in fluorescence throughout the secretory pathway. Non specificexpression patterns are not amenable for use as a FRET donor because alarge percentage of the emitted light originates from irrelevantcellular locations and results in a high background, which precludeslarge signal changes and may even be a source of artifactualfluorescence changes. We have found that N-terminal fusion of a ˜20amino acid plasma membrane targeting sequence from Lyn tyrosine proteinkinase (Resh. (1994) Cell 76 (3) 411–3), a member of the Src family oftyrosine protein kinases, and C-terminal fusion of CAAX motifs (Mageeand Marshall (1999) Cell 98 9–12) to GFP results in specificfluorescence at the plasma membrane of transfected mammalian cells.

Example XI—Transient Transfections and Generation of Stable Cell Lines

The targeted naturally fluorescent protein constructs were transientlytransfected into CHO cells using the lipid-mediated transfection reagentLipofectamine (GibcoBRL) at a 1:12 DNA to lipid ratio. Expression of themembrane targeted sapphire GFP in rat basophilic leukemia (RBL-1) cellswas achieved by transfecting wild-type RBL cells with the GFP constructsby electroporation. Cells lines stably expressing the GFP constructswere generated for both RBL-1 and CHO host cells by selecting forgeneticin (G418) resistance. Targeted GFP-expressing clones were sortedusing a Becton Dickinson FACS Vantage SE cell sorter equipped with aCoherent Innova Krypton 302 laser. Sapphire GFP was excited using the407 nm line of the krypton laser. Individual cells were sorted intomicrotiter plates based on fluorescence through a 530/30 nm emissionfilter.

Example XII—Measurement of Membrane Potentials Using Targeted NaturallyFluorescent Proteins

Trimethine oxonol Loading.(bis-(1,3-dialkyl-2-thiobarbiturate)(DiSBAC_(x)(3)) acceptors, where xrefers to the number of carbons in alkyl substituents, were loaded intowashed cells by incubation at room temperature for 30 minutes withgentle shaking. RBL cells were harvested from confluent flasks usingnon-enzymatic cell dissociation buffer (GibcoBRL), pelleted, washed inBath1 buffer (160 mM NaCl, 4.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mMD-glucose, 10 mM HEPES pH 7.4), resuspended to 2×10⁶ cells/mL in Bath1containing oxonol, and incubated at room temperature for 30 minutes withgentle shaking. Oxonol was washed out and cells were plated at 1×10⁵cells per well in poly-L-lysine coated black-wall microtiter plates(Costar). Cells were allowed to attach to the poly-L-lysing by settlingfor 30 minutes or centrifugation in the plate at 170×g for 5 min. Thebutyl oxonols were loaded using 0.2 mg/mL pluronic 127 and the hexylwith pluronic and 1.5 mM β-cyclodextrin. Both butyl and hexyl oxonolswere washed out before assaying cells. The ethyl oxonols were loadedwithout pluronic and left in the buffer during the assays.

Addition of trimethine oxonols to CHO or RBL-1 cells expressingLyn-Sapphire GFP caused an approximately 10–50% quenching of the GFPfluorescence and a concomitant increase in the oxonol emission. Thespectral changes are indicative of FRET between the plasma membranetargeted GFP and the membrane-bound oxonol. At normal negative cellularresting membrane potentials the majority of oxonol acceptors arelocalized in the outer plasma membrane leaflet and only partialquenching of the GFP donor is expected. Direct excitation of the FRETacceptor is a common problem in FRET applications. For this reason, wedecided to implement Sapphire GFP since it has a single excitation peakat 395 nm, the excitation minimum for trimethine oxonols. Even thoughS65T mutants have ˜2-fold greater extinction coefficients, the ˜490 nmexcitation light necessary to optimally excite these mutants results insubstantial direct excitation of oxonol, which causes non FRET emission.The oxonol excitation spectrum has a minimum at ˜400 nm which is idealfor Sapphire excitation. At 490 nm, the excitation maximum of S65Tmutants, the oxonol is ˜15% excited compared its maximum. Since theoxonol has a maximal extinction of ˜200,000 M⁻¹ cm⁻¹ this corresponds toan effective extinction of 30,000 M⁻¹ cm⁻¹ which is comparable to GFPs.Oxonol molecules not located at the plasma membrane are not efficientlyexcited with 405 nm light, which is ˜130 nm shorter than the oxonolabsorbance maximum. Furthermore, oxonols not in membranes areessentially non fluorescent. Taken together, the light emitted from thestained cells is primarily from the plasma membrane where the Sapphiredonors are located. Measurement of fluorescence emission from stainedcells on coverslips, in cuvettes, and in microtiter plates indicatedthat the 5–40% of the detected oxonol emission originates from directexcitation. The percentage depends on which oxonol is used, sincebinding to different cellular membrane varies with dye physiochemicalproperties. The more hydrophilic and water-soluble oxonols easilypartition into most intracellular membranes and compartments.

Calibration of Voltage Sensitivity

We evaluated the voltage-sensitivity of the GFP/trimethine oxonol FRETpairs using whole-cell voltage clamp techniques to control the voltageacross the plasma membrane while simultaneously recording theintensities of the GFP and oxonol emissions.

Simultaneous electrical and optical measurements were performed on aZeiss 100 inverted epifluorescence microscope adapted for simultaneousemission ratioing. Experiments were conducted with either a 40×1.4 NA or63×1.25 NA Zeiss Fluar objective. The excitation source was a 75 W xenonarc lamp. Excitation light was passed through a 405 nm±10 nm filter(Omega Optical) and was used to excite cells expressing the naturallyfluorescent proteins after passage through a 425 nm mirror. Thefluorescence emission was passed through a 435 nm long pass filterbefore being split with a 550 nm DRLP second dichroic. GFP emissionmeasurements were made after being filtered through 510±30 nm filter.The transmitted oxonol emission was measured after passage through a 740nm dichroic and a 580±35 nm band pass filter. The emitted light wassimultaneously detected using two PMTs (Hamamatsu). Infrared light1000±150 nm was used to image the cell for patch clamping. Cells werestained with 6 uM DiSBAC₂ for a minimum of 20 minutes unless otherwisenoted.

Patch Clamping

All patch clamp experiments were performed at room temperature using anAxoPatch 1C amplifier and the pClamp software suite (Axon Instruments,Inc. Foster City, Calif.). A ground was created by placing an Ag/AgClpellet placed directly in the bath. Patch pipettes were made on aFlaming/Brown micropipette puller (Model P-97, Sutter Instruments, Inc.Novato, Calif.) from borosilicate glass with resistances when filledwith pipette solution of 3–6 MΩ. The pipette solution was composed of140 mM K-gluconate, 2 mM MgCl₂, 1 mM CaCl₂, 10 mM EGTA, 10 mM HEPES andpH 7.2.

An example of a large voltage-sensitive FRET response from a single cellis shown in FIG. 25. Upon depolarization, the green donor emissiondecreases and the oxonol emission increases as predicted for a netoxonol translocation from the outer plasma membrane leaflet to theinner, as depicted in FIG. 24. These fluorescence changes are inopposite directions to those observed using a coumarin-lipid donor,which binds to the extracellular plasma membrane leaflet (Gonzalez andTsien, (1997) Chem. Biol. 4 (4) 269–77). These data confirm thetargeting of the naturally fluorescent protein to the inner leaflet ofthe plasma. For the coumarin-lipid/oxonol pairs the majority of thesignal can be manipulated by changing the relative dye stainingconcentrations. In the GFP/oxonol case the majority of the signal changeis almost always from GFP, probably because of the spill over of the GFPemission into the oxonol signal.

Example XIII—Comparison of Different Membrane Targeting Sequences

We systematically characterized and compared the voltage-sensitivity of6 Sapphire GFP constructs based on the N-terminal fused Lyn domain andthe C-terminal fused-CAAX motifs. From these experiments we wanted todetermine the generality of voltage-sensitive FRET with different GFPdonors and also to find a probe with intrinsically greatervoltage-sensitivity. The six constructs are listed in Table 4. Therationale for choosing these constructs was that by modifying thetargeting sequence length, attachment point, and charge, we had a goodchance of modulating FRET, since it is typically sensitive to thedistance and orientation between donor and acceptor chromophores.Truncations of the Lyn domain were prepared to test whether shorteningthe targeting sequence would retain membrane binding and if so whetherthe shorter linker placed the GFP closer to the membrane and resulted ingreater FRET, and possibly voltage-sensitive FRET. Two truncations ofthe targeting sequence were made at aspartic acid residue 10 (D10Sapphire) and aspartic acid residue 15 (D15 Sapphire). Also, a triplearginine patch present in the N-terminal region of Src tyrosine proteinkinase was substituted for the corresponding Lyn residues in the LynSapphire construct (Lyn RRR Sapphire). Two other constructs were made byadding a C-terminal plasma membrane targeting sequence from K-Ras; onewith the C-terminal anchor fused to the Sapphire GFP and the other alsoconsisting of the Lyn sequence fused to the N-terminus, a construct withtwo membrane anchors. The C-terminal constructs were prepared to test ifmembrane anchoring via the C-terminal would cause a different FRETinteraction with the oxonol. In the case of the double-anchored probe,it was speculated that such a major modification might lead to anorientation change that could modulate FRET. A third factor that wasvaried was the number of positive charges on the peptide linker. Thisissue was addressed, in part, by the triple arginine and the K-RASconstructs that contain a large number of positively charged aminoacids. Since the oxonols are negatively charged, the possibility of aweak electrostatic attraction between donor and acceptor wastantalizing. Finally, the number of positive charges on the innerleaflet could shift the voltage dependence of the oxonol displacementcurrents to more negative voltages, which would move thevoltage-sensitivity maximum into a more physiologically relevant range,more negative of −5 mV, which was previously observed in LmTK⁻ cells(Gonzalez and Tsien, (1995) Biophys. J. 69 (4) 1272–80).

The constructs were transiently transfected into CHO cells andvoltage-sensitivity was determined according to the procedure describedabove, using a minimum of 5 cells for each construct. Data from 3separate runs were averaged for the hyperpolarization and depolarizationprotocol shown in the inset. The measured sensitivities were similar forthe two different voltage pulses. Cells with above average brightnessand apparent membrane targeting were selected for patch-clamping. Cellcapacitance values were typically between 10 and 15 pF. The results aresummarized in Table 5.

TABLE 5 Construct Name Ratio Change per 100 mV Lyn Sapphire 34 +/− 5 LynD15 Sapphire 21 +/− 8 Lyn D10 Sapphire 29.5 +/− 7   Lyn RRR Sapphire 26+/− 7 Ras Sapphire 59 +/− 8 LynRas Sapphire  15 +/− 10

The first major observation was that all the constructs resulted involtage-sensitive FRET. This is important because it demonstrates thatvoltage-sensitive FRET is generally applicable as long as the GFP isspecifically targeted and close enough to the membrane to undergo FRETwith the oxonol.

Secondly, it is clear from the results that the RAS targeted SapphireGFP was nearly 2-fold more sensitive than the other constructs underthese transient conditions. No fluorescence changes were observed in theabsence of added oxonol. The 60% ratio change per 100 mV seen for theRAS Sapphire GFP donor is comparable to the high sensitivity seen withpurely exogenous probes that use coumarin phospholipid donors. Theresults demonstrate that the nature of the targeting sequence caninfluence the magnitude and sensitivity of the fluorescencemeasurements, even among very similar localization sequences.

Example XIV—Temporal Analysis of Different Fluorescent Protein OxonolPairs

The temporal response of the GFP/oxonol FRET change is dependent on theoxonol translocation rate and follows the previously observed trend withoxonol acceptors, faster responses with more hydrophobic acceptors. Themeasured time constants for the ethyl and butyl oxonol acceptors are ˜50and ˜5 ms respectively in the targeted GFP expressing cells. The FRETresponse and the electrophysiologically measured displacement currentshave identical time constants. The measured response times are 2–4 foldfaster than previously reported and may be a property of the cells usedor from a slight difference in temperature. The temporal responses ofDiSBAC₄ displacement currents in cells expressing GFP and those of thehost cells did not show any significant differences.

Example XV—Measurement of Voltage Changes in High Throughput ScreeningFormat

Fluorescence measurements from 96 well mircoplates were made with theVIPR™ microtiter plate reader (U.S. application Ser. No. 09/122,544,filed Jul. 24, 1998 entitled “Detector and Screening Device for IonChannels.”)

Cells were illuminated with light from a 300 W xenon arc lamp passedthrough a 400±7 nm band pass excitation filter and fluorescence emissionintensities were recorded via two PMTs using a 535±18 nm band passemission filter to collect sapphire GFP emission and a 580±30 nm bandpass filter to collect trimethine oxonol emission.

A typical VIPR™ run involved recording fluorescence intensities at thedonor and acceptor wavelengths simultaneously for 35 seconds at 1 Hzwith liquid addition occurring between 12 and 15 seconds. Data wereanalyzed by first subtracting background, (intensities from wellswithout cells) from the sample wells. The fluorescence ratio (oxonol/GFPemission) was then calculated and normalized to the starting ratio ofeach well before liquid addition. The final ratio was determined usingdata between 17 and 24 seconds. The fraction of GFP emission thatemitted in the wavelength range used for oxonol detection (580 nm±30)was determined using cells prior to the addition of the oxonol. Theleakage of the GFP emission into the oxonol signal was subtracted priorto data analysis, for oxonol stained cells assuming the same GFPemission profile in the presence of oxonol.

The homogeneity of the stable lines enabled us to perform membranepotential assays using an integrated liquid handling and fluorescenceplate reader capable of fast simultaneous emission ratioing. Cellularassays that test for compounds that block high K⁺ induceddepolarizations have been successfully performed in microtiter platesusing all the stable Sapphire GFP cell lines with trimethine oxonolsFRET acceptors. The RBL-1 cells have an endogenous inward rectifying K⁺channel, IRK, that sets the membrane potential to the K⁺ equilibriumpotential. Addition of a high K⁺ solution (164.5 mM KCl, 2 mM CaCl₂, 1mM MgCl₂, 10 mM D-glucose, 10 mM HEPES pH 7.4) causes a rapiddepolarization that is detected with the FRET membrane potential probes.Data for the hexyl trimethine oxonol in the Lyn RBL line is shown inFIG. 26.

The data shows very reproducible well-to-well responses, with the timeresponse limited by the liquid addition. The ability to use microtiterplates makes the targetable voltage probes compatible withhigh-throughput drug screening and allows facile optimization ofacceptor concentration for the best ratio response. The utility forcompound screening is further demonstrated by incubating the cells withBa²⁺ which blocks IRK and depolarizes the cells. This causes the high K⁺signal to be blocked since the cells are already depolarized when thehigh K⁺ solution is added. The Ba²⁺ dose-response in the Lyn SapphireGFP/butyl trimethine oxonol is shown in FIG. 27. Microtiter wellstreated with the IRK antagonists can be clearly identified.

The foregoing invention has been described in some detail by way ofillustration and example, for purposes of clarity and understanding. Itwill be obvious to one of skill in the art that changes andmodifications may be practiced within the scope of the appended claims.Therefore, it is to be understood that the above description is intendedto be illustrative and not restrictive. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to thefollowing appended claims, along with the full scope of equivalents towhich such claims are entitled.

All patents, patent applications and publications cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each individual patent, patentapplication or publication were so individually denoted.

1. A composition comprising: a) a coumarin-labeled phospholipid havingthe formula:

wherein R is a hydrophobic lipid anchor comprising 14–35 carbons; and b)a mobile hydrophobic molecule capable of redistributing from a firstface of a cellular membrane to a second face of the cellular membrane inresponse to a change in the membrane potential, and which can undergoenergy transfer with the coumarin-labeled phospholipid upon exposurewith excitation light by either (i) donating excited state energy to thecoumarin-labeled phospholipid, or (ii) accepting excited state energyfrom the coumarin-labeled phospholipid.
 2. The composition of claim 1,wherein said mobile hydrophobic molecule comprises a polymethine oxonol.3. The composition of claim 2, wherein said mobile hydrophobic moleculeis selected from DiSBA-C₆-(3), DiSBA-C₆-(5) or DiSBA-C₁₀-(3).
 4. Thecomposition of claim 3, wherein said mobile hydrophobic molecule isDiSBA C₆-(3).
 5. A kit comprising in separate vessels: a) acoumarin-labeled phospholipid having the formula:

wherein R is a hydrophobic lipid anchor comprising 14–35 carbons; and b)a mobile hydrophobic molecule capable of redistributing from a firstface of a cellular membrane to a second face of the cellular membrane inresponse to a change in the membrane potential, and which can undergoenergy transfer with the coumarin-labeled phospholipid upon exposurewith excitation light by either (i) donating excited state energy to thecoumarin-labeled phospholipid, or (ii) accepting excited state energyfrom the coumarin-labeled phospholipid.
 6. The kit of claim 5, whereinsaid mobile hydrophobic molecule comprises a polymethine oxonol.
 7. Thekit of claim 6, wherein said mobile hydrophobic molecule is selectedfrom DiSBA-C₆-(3), DiSBA-C₆-(5) or DiSBA-C₁₀-(3).
 8. The kit of claim 7,wherein said mobile hydrophobic molecule is DiSBA C₆-(3).
 9. A method ofdetecting a change in the membrane potential in at least one cell,comprising: a) contacting said at least one cell with a mobilehydrophobic molecule, wherein said mobile hydrophobic moleculeredistributes from a first face of the membrane to a second face of themembrane in response to said change in the membrane potential, and whichundergoes energy transfer with a coumarin-labeled phospholipid uponexposure with excitation light by either (i) donating excited stateenergy to the coumarin-labeled phospholipid, or (ii) accepting excitedstate energy from the coumarin-labeled phospholipid, wherein theefficiency of energy transfer between said mobile hydrophobic moleculeand said coumarin-labeled phospholipid is dependent on the membranepotential, said coumarin-labeled phospholipid having the formula:

wherein R is a hydrophobic lipid anchor comprising 14–35 carbons; b)contacting said at least one cell with said coumarin-labeledphospholipids, c) exposing the membrane to a stimulus thereby alteringthe membrane potential, d) exposing the membrane to excitation light, e)determining the degree of energy transfer between the mobile hydrophobicmolecule and the coumarin-labeled phospholipid, f) relating the degreeof energy transfer to the membrane potential thereby detecting a changein the membrane potential.
 10. A method of identifying a test chemicalthat affects a change in membrane potential in at least one cellcomprising the steps of: a) contacting said at least one cell with amobile hydrophobic molecule, wherein said mobile hydrophobic moleculeredistributes from a first face of the membrane to a second face of themembrane in response to said change in the membrane potential, and whichundergoes energy transfer with a coumarin-labeled phospholipid uponexposure with excitation light by either (i) donating excited stateenergy to the coumarin-labeled phospholipid, or (ii) accepting excitedstate energy from the coumarin-labeled phospholipid, wherein theefficiency of energy transfer between said mobile hydrophobic moleculeand said coumarin-labeled phospholipid is dependent on the membranepotential, said coumarin-labeled phospholipid having the formula:

wherein R is a hydrophobic lipid anchor comprising 14–35 carbons; b)contacting said at least one cell with said coumarin-labeledphospholipids, c) exposing the membrane to said test chemical therebyaltering the membrane potential, d) exposing the membrane to excitationlight, e) determining the degree of energy transfer between the mobilehydrophobic molecule and the coumarin-labeled phospholipid, f) relatingthe degree of energy transfer to the membrane potential therebyidentifying said test chemical.
 11. The method of claim 9 or 10, whereinsaid mobile hydrophobic molecule comprises a polymethine oxonol.
 12. Themethod of claim 11 or 10, wherein said mobile hydrophobic molecule isselected from DiSBA-C₆-(3), DiSBA-C₆-(5) or DiSBA-C₁₀-(3).
 13. Themethod according to claim 12 or 10, wherein said mobile hydrophobicmolecule is DiSBA-C₆-(3).
 14. The method according to claim 9 or 10,wherein said membrane potential is mediated by an ion channel.
 15. Thecomposition of claim 1, wherein said coumarin-labeled phospholipid hasthe formula:


16. The composition of claim 1, wherein said mobile hydrophobic moleculeis a tetraaryl borate or a lanthanide complex.
 17. The composition ofclaim 1, wherein said mobile hydrophobic molecule is capable ofredistributing from a first face of a cellular membrane to a second faceof the cellular membrane in response to a change in the membranepotential with a time constant of less than about 10 milliseconds. 18.The method of claim 9 or 10, wherein said mobile hydrophobic moleculeredistributes from a first face of the membrane to a second face of themembrane in response to a change in the membrane potential with a timeconstant of less than about 5 milliseconds.
 19. The method of claim 9,wherein said stimulus is an electrical stimulus.
 20. The method of claim9 or 10, wherein said coumarin-labeled phospholipid has the formula:


21. The method of claim 9 or 10, wherein said mobile hydrophobicmolecule is a tetraaryl borate or a lanthanide complex.
 22. The kit ofclaim 6, wherein said coumarin-labeled phospholipid has the formula:


23. The kit of claim 6, wherein said mobile hydrophobic molecule is atetraaryl borate or a lanthanide complex.
 24. The kit of claim 6,wherein said mobile hydrophobic molecule is capable of redistributingfrom a first face of a cellular membrane to a second face of thecellular membrane in response to a change in the membrane potential witha time constant of less than about 10 milliseconds.